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Mechanisms of anaesthetic depression of neocortical arousal El-Beheiry, Hossam El-Dean Mohamed 1990

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MECHANISMS OF ANAESTHETIC DEPRESSION OF NEOCORTICAL AROUSAL by HOSSAM EL-DEAN MOHAMED EL-BEHEIRY M.B, Ch.B., Cairo Univers ity, 1978 M.Sc. (Anaesthesia), Cairo Univers i ty, 1982 M.D. (Anaesthesia), Cairo University 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FAULTY OF GRADUATE STUDIES Department of Pharmacology & Therapeutics, Faculty of Medicine We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1990 © HOSSAM EL-BEHEIRY, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmacology and Therapeutics The University of British Columbia Vancouver, Canada Date 28 dune 1990 DE-6 (2/88) i i ABSTRACT The most w ide ly accepted hypotheses suggest t h a t general a nae s t he t i c s i n t e r r u p t consc ious processes i n the b r a i n by decreas ing s ynapt i c e x c i t a t i o n or by p o t e n t i a t i n g s ynapt i c i n h i b i t i o n , e s p e c i a l l y i n the neocortex. The p u t a t i v e t r a n s m i t t e r s i n the n e u r o l o g i c a l systems that generate n e o c o r t i c a l arousa l i n c l ude a c e t y l c h o l i n e , glutamate and T - aminobuty ra te (GABA). The pr imary o b j e c t i v e here was to determine the neuronal mechanisms by which anae s t he t i c s may obtund t h i s a r o u s a l . The m a j o r i t y of the i n v e s t i g a t i o n s were c a r r i e d out on pyramidal neurons i n l a y e r s IV and V of guinea p i g neocortex ( i n v i t r o s l i c e s ) , u s ing i n t r a -c e l l u l a r record ing and pharmaco log i ca l , i n c l u d i n g m i c r o i o n t o p h o r e t i c , techn iques . Bath a p p l i c a t i o n s of s t r u c t u r a l l y d i s s i m i l a r a n a e s t h e t i c s , i s o f l u r a n e - a halogenated e t he r , and A l t h e s i n - a s t e r o i d a l p r e p a r a t i o n , i n c oncen t r a t i on s of 0.5-2.5 minimum a l v e o l a r concen t r a t i on (MAC) and 10-1300 nM, r e s p e c t i v e l y , produced a small h y p e r p o l a r i z a t i o n (3-5 mV) which was a s s oc i a t ed with an inc rease i n input conductance (10-30%). The lower c oncen t r a t i o n s (0.5-1.5 MAC and 10-200 LIM) of these agents which are most r e l e v a n t to the p roduc t i on of unconsciousness d i d not s i g n i f i c a n t l y a f f e c t the pas s i ve membrane p r o p e r t i e s . However, they produced s t r i k i n g decreases i n spontaneous a c t i v i t i e s and the r e p e t i t i v e sp ike f i r i n g evoked by orthodromic ( e l e c t r i c a l ) s t i m u l a t i o n or i n t r a c e l l u l a r c u r r en t i n j e c t i o n s . Because the observed changes i n membrane p r o p e r t i e s cou ld not e x p l a i n the r educ t i on i n neuronal e x c i t a b i l i t y , the e f f e c t s of anae s the t i c s were i n v e s t i g a t e d e x t e n s i v e l y on e x c i t a t o r y and i n h i b i t o r y po s t s ynap t i c p o t e n t i a l s (EPSPs and IPSPs). The a p p l i c a t i o n of i s o f l u r a n e or A l t h e s i n induced a dose-dependent, r e v e r s i b l e depres s ion i n the ampl i tude of EPSPs, i i i w i th E C 5 Q s of 1 MAC and ~50 uM, r e s p e c t i v e l y . The IPSPs a l s o were reduced i n a dose-dependent manner. In order to e l i m i n a t e p o s s i b l e shunt ing of the EPSPs by the GABA-act ivated Cl-conductance t h a t produces the IPSP i n the observed EPSP-IPSP sequence, a GABA A -antagon i s t , b i c u c u l l i n e , was add i -t i o n a l l y a p p l i e d . De sp i te t h i s IPSP-blockade, the a n a e s t h e t i c s s t r o n g l y depressed the EPSPs as we l l as e p i l e p t i f o r m a c t i v i t i e s evoked by subp ia l e l e c t r i c a l s t i m u l a t i o n . In cognizance of the p o s s i b i l i t y t h a t a po s t s ynap t i c a t t e n u a t i o n of respons iveness to t r a n s m i t t e r substances may be i n vo l ved i n the EPSP depres s i on , the neuronal s e n s i t i v i t i e s to a c e t y l c h o l i n e , g lutamate. and GABA were determined. Anae s t he t i c a d m i n i s t r a t i o n markedly reduced the d e p o l a r i z a t i o n s and a s s oc i a t ed conductance changes evoked by d e n d r i t i c a p p l i c a t i o n s of a c e t y l c h o l i n e , glutamate and N-methy1-D-aspartate (NMDA). The h y p e r p o l a r i z i n g responses t o somatic a p p l i c a t i o n s of GABA were not a f f e c t e d s i g n i f i c a n t l y whereas the d e p o l a r i z i n g e f f e c t s observed w i th i t s d e n d r i t i c a p p l i c a t i o n were s l i g h t l y depressed. Seme degree of s e l e c t i v i t y a l s o was ev ident from the lower E C ^ s f o r the i s o f l u r a n e - and A l t h e s i n -induced depress ions of responses to a c e t y l c h o l i n e compared w i t h g lutamate. Under i n v i t r o c o n d i t i o n s of hypomagnesia the responses to a c e t y l c h o l i n e were t o t a l l y b locked and the order of depress ion i n the responses t o GABA and glutamate was r e ve r s ed ; t h i s may be of importance i n the mechanism f o r the known inc rease i n anae s the t i c requirements i n c l i n i c a l syndromes a s soc i a ted w i th hypomagnesaemia. 2+ Because the genes i s of s ynapt i c t r a n s i e n t s i s a f f e c t e d by Ca i n f l u x o r d i s p o s i t i o n , the i n t e r a c t i o n s of anaes the t i c s were i n v e s t i g a t e d on sp i ke a f t e r h y p e r p o l a r i z a t i o n s (AHPs). The AHPs which are produced s p e c i f i c a l l y by 2+ + a Ca - a c t i v a t e d K -conductance were suppressed by the anae s t he t i c s i n a IV dose-dependent manner under conditions where contaminating IPSPs had been blocked by bicuculline. Since the passive membrane properties were unaffected, an interference with a transmembrane Ca +-influx may be involved in the anaesthetic actions. The effects of anaesthetics on glutamate-induced and voltage-dependent 2+ 2+ increases in intraneuronal Ca ([Ca ].) were determined in cultured hippocampal neurons with a Ca-sensitive probe (Fura-2) and microspectro-fluorometric techniques. Isoflurane application depressed the increases in 2+ [Ca ]. produced by application of glutamate under conditions where its actions would be favoured at NMDA- and quisqualate-subtypes of receptors. + 2+ K -induced increases in [Ca ]•• also were reduced by application of isoflurane, probably due to actions on voltage-dependent Ca-channels in the membrane. These investigations have provided evidence for the f i r s t time that excitatory transmitter actions in neocortex are selectively depressed by anaesthesia. A plausible mechanism would include suppression of the postsynaptic Ca-conductances associated with the AHPs and glutamatergic, as well as cholinergic interactions at pre- and post-synaptic sites on neurons involved in neocortical arousal. V TABLE OF CONTENTS CHAPTER Page TITLE PAGE 1 ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES x LIST OF FIGURES x i ACKNOWLEDGEMENT xv LIST OF ABBREVIATIONS xv i DEDICATION x v i i 1 INTRODUCTION 1 1.1 General scope of t o p i c 1 1.2 The consc ious system of the b r a i n 2 1.2.1 D e f i n i t i o n s 2 1.2.2 I n t e g r a t i on of f u n c t i o n and a c t i v i t y of the consc ious subsystems 3 1.2.3 Func t i ona l neuroanatomy of the consc iousness system 3 1.2.4 N e o c o r t i c a l a c t i v a t i o n 6 1.2.5 A c t i v a t i n g t r a n s m i t t e r s i n the neocortex 7 1.2.6 The a c t i v a t i n g t r a n s m i t t e r s and t h e i r i o n i c mechanisms of a c t i o n 8 1.2.6.1 A c e t y l c h o l i n e 8 1.2.6.2 S-Glutamate 11 1.3 The anae s the t i c s t a t e 13 1.3.1 H i s t o r i c a l i n t r o d u c t i o n and d e f i n i t i o n s 13 1.3.2 S i t e of a c t i o n of anae s the t i c s i n the consc iousness system 14 1.3.3 Mechanisms of a c t i on of general anae s the t i c s 16 1.3.3.1 N o n - s p e c i f i c ( l i p i d ) hypotheses 16 1.3.3.2 S p e c i f i c ( p r o t e i n ) hypotheses 20 1.3.3.3 Anae s the t i c mo lecu la r mechanisms and c e l l u l a r e x c i t a b i l i t y 23 1.3.3.4 Anae s the t i c a c t i o n s on cy top la smic membrane s i t e s 25 - v i -CHAPTER Page 1.4 Hypotheses and Ob jec t i ve s 27 2 METHODS 27 2.1 Animals 27 2.2 S l i c e p repa ra t i on 28 2.3 S o l u t i o n s and drugs 29 2.4 I n t r a c e l l u l a r r eco rd ing arrangement 31 2.5 Computer a n a l y s i s 33 2.6 I on tophore t i c techniques 35 2.6.1 E l e c t r ode s 35 2.6.2 S o l u t i o n s and equipment 36 2.6.3 Exper imental procedures 37 2.7 I n t r a c e l l u l a r C a 2 + measurement 37 2.7.1 Neuronal c u l t u r e s 37 2.7.2 Storage and load ing of Fura-2 AM 38 2.7.3 I n t r a c e l l u l a r C a 2 + measurements 38 2.7.4 C a l i b r a t i o n of the system 40 2.8 S t a t i s t i c a l a n a l y s i s 40 3 SPONTANEOUS ACTIVITY, REPETITIVE FIRING AND PASSIVE MEMBRANE PROPERTIES 40 3.1 Re su l t s 40 3.1.1 E f f e c t s on spontaneous a c t i v i t y and evoked r e p e t i t i v e f i r i n g . 41 3.1.2 E f f e c t s on Vm and membrane e l e c t r i c a l p r o p e r t i e s 43 3.2 D i s cu s s i on 49 4 EFFECTS ON SYNAPTIC TRANSIENTS 50 4.1 Re su l t s 50 4.1.1 Rest ing membrane p r o p e r t i e s 50 -VI1-CHAPTER Page 4.1.2 Dose-response depress ion of EPSPs 51 4.1.3 EPSP a t t enua t i on i n the presence of IPSP-blockade 54 4.1.4 E f f e c t s on o r t h o d r o m i c a l l y and i n t r a c e l l u l a r ^ evoked sp ikes 58 4.1.5 Dose-response r e l a t i o n s h i p f o r IPSP suppress ion dur ing Cs-blockade of the K-conductances 58 4.2 D i s cu s s i on 61 4.2.1 Anaes the t i c depress ion of EPSPs 65 4.2.2 Anaes the t i c a c t i o n s on the IPSPs 68 5 ANAESTHETIC INDUCED ALTERATIONS IN NEURONAL RESPONSIVENESS TO ACTIVATING TRANSMITTERS AND RELATED SUBSTANCES 70 5.1 Resu l t s 70 5.1.1 Cont ro l responses to i o n t o p h o r e t i c a l l y a pp l i ed agents 70 5.1.1.1 A c e t y l c h o l i n e 70 5.1.1.2 Glutamate and N-methyl -D-aspartate 72 5.1.1.3 Gamma-aminobutyrate and bac lo fen 74 5.1.2 Anaes the t i c induced a l t e r a t i o n s i n responses to e x c i t a t o r y and i n h i b i t o r y t r a n s m i t t e r substances 81 5.1.2.1 Responses to ACh, Glu and NMDA 81 5.1.2.2 Responses t o GABA 87 5.1.3 S e l e c t i v i t y i n the depress ion of responses to t r a n s m i t t e r substances 87 5.1.3.1 Anae s the t i c i n t e r a c t i o n s w i th t r a n s m i t t e r s 87 5.1.3.2 Time course of anae s the t i c ac t i on s 93 5.1.3.3 Dose-resposne r e l a t i o n s h i p s 93 5.1.3.4 Anae s the t i c e f f e c t s on the p o t e n t i a t i o n of G lu - responses by ACh 97 5.2 D i s cus s i on 97 5.2.1 Responses to t r a n s m i t t e r and r e l a t e d agents 97 5.2.1.1 A c e t y l c h o l i n e 101 5.2.1.2 Glutamate and N-methyl -D-aspartate 102 5.2.1.3 Y -aminobuty ra te 102 5.2.2 Anae s the t i c induced a l t e r a t i o n s i n the c h e m o s e n s i t i v i t y of e x t r a c e l l u l a r l y a p p l i e d t r a n s m i t t e r agents 104 6 EFFECTS OF HYPOMAGNESIA ON TRANSMITTER AND ANAESTHETIC ACTIONS 109 6.1 Re su l t s 109 6.1.1 E f f e c t s of bath a p p l i c a t i o n of Mg-free ACSF 110 - v i i i -CHAPTER Page 6.1.2 E f f e c t s of gradual removal Of [Mg 2 + ] 0 110 6.1.3 E f f e c t s of i n c r e a s i n g [Mg 2 + ] 114 6.1.4 E f f e c t s of ex te rna l C a 2 + and Mg 2 + e x c l u s i o n on the Glu responses 119 6.1.5 Anae s the t i c a c t i on s dur ing Mg-free pe r f u s i on 119 6.2 D i s cu s s i on 123 6.2.1 Suppress ion of the ACh-induced responses 123 6.2.2 Changes i n [ M g 2 + ] 0 do not s i g n i f i c a n t l y a f f e c t G lu - i nduced responses 124 6.2.3 GABA-depo la r i za t i on s are c o n s i s t e n t l y a t tenuated by the removal of [ M g 2 + ] 0 125 6.2.4 Enhancement of neuronal e x c i t a b i l i t y by the removal of [ M g 2 + ] 0 126 6.2.5 Anae s the t i c - i nduced depress ion of Glu and GABA responses i n Mg 2 + - f r ee media 127 7 ANAESTHETIC-INDUCED ATTENUATION OF POSTSPIKE AHP MEDIATED BY Ca-ACTIVATED K-CONDUCTANCE 127 7.1 Resu l t s 127 7.1.1 Depress ion of AHPs 128 7.1.2 B i c u c u l l i n e - b l o c k a d e of i n h i b i t o r y po s t s ynap t i c p o t e n t i a l s 133 7.2 D i s cu s s i on 133 7.2.1 Mechanism(s) of anaes the t i c i n t e r f e r e n c e wi th AHP 137 7.2.2 S i g n i f i c a n c e of AHP i n h i b i t i o n i n n e o c o r t i c a l neurons generat ion 137 8 EFFECTS OF ISOFLURANE ON THE GLUTAMATE- AND POTASSIUM-INDUCED INCREASES IN INTRANEURONAL CALCIUM CONCENTRATION 138 8.1 Resu l t s 138 8.1.1 Responses to ACh and carbachol 139 8.1.2 E f f e c t s of i s o f l u r a n e on r e s t i n g [Ca^l^ and Glu a c t i on s 139 8.1.3 I s o f l u r ane a c t i on s on K-evoked inc reases i n [ C a 2 + ] j 144 8.2 D i s cus s i on 144 9 GENERAL DISCUSSION 147 CHAPTER Page 9.1 N e o c o r t i c a l u n i t a c t i v i t y and general anaes thes i a 148 9.2 Anae s the t i c a c t i o n s : depress ion of e x c i t a t i o n or p o t e n t i a t i o n of i n h i b i t i o n ? 148 9.3 The anae s the t i c s t a te and suppress ion of consc iousness mechanisms 149 9.4 I on i c mechanisms of anaes the t i c a c t i o n 152 9.5 Mo lecu l a r mechanisms of anaes thes ia — the l i p i d / p r o t e i n cont rover sy 155 10 SUMMARY AND CONCLUSIONS 156 11 REFERENCES 160 -X-LIST OF TABLES TABLE Page 1 Anaes the t i c depress ion of neuronal responses to t r a n s m i t t e r substances 91 2 E f f e c t s of Mg-free media on sensor imotor neurons 113 3 E f f e c t s of i s o f l u r a n e on [Ca2 + ] 1 - i n c rea se s evoked by g lutamate and K + 143 - x i -LIST OF FIGURES FIGURE Page 1 Anaes the t i c d e l i v e r y system and reco rd ing chamber used i n these i n v e s t i g a t i o n s . 30 2 Diagram showing the n e o c o r t i c a l s l i c e and s i t e s of r e co rd i n g and s t i m u l a t i o n and types of i on topho re t i c e l e c t r ode s used. 32 3 O rgan i za t i on of exper imenta l set-up f o r data d i s p l a y , a c q u i s i t i o n and r e t r i e v a l . 34 4 I s o f l u rane and A l t h e s i n induced depress ion of spontaneous sp ike a c t i v i t i e s . 42 5 Anae s the t i c - i nduced inc reases i n the t h r e s h o l d f o r sp ike genes i s . 44 6 Anaes the t i c i n t e r f e r e n c e w i th the r e p e t i t i v e f i r i n g a b i l i t i e s of n e o c o r t i c a l neurons. 46 7 A l t e r a t i o n s i n the pa s s i ve membrane p r ope r t i e s by a p p l i c a t i o n of i s o f l u r a n e and A l t h e s i n . 47 8 E f f e c t s of i s o f l u r a n e on the input r e s i s t a n c e of an a n t e r i o r c i n g u l a t e neuron. 48 9 EPSP-depress ion induced by i s o f l u r a n e and A l t h e s i n 52 10 Dose-dependent suppress ion of the ampl itude and r a t e o f decay by i s o f l u r a n e . 53 11 A l t h e s i n - i n d u c e d dose-dependent depress ion of the ampl i tudes and ra te s of decay of n e o c o r t i c a l evoked EPSPs. 55 12 E p i l e p t o g e n i c a c t i v i t y and EPSP supress ion by anaes the t i c a p p l i c a t i o n s i n the presence of GABA-ergic blockade i n n e o c o r t i c a l neurons. 56 13 Anaes the t i c i n t e r f e r e n c e w i t h the s y n a p t i c a l l y - i n d u c e d sp ikes and d i r e c t l y - e v o k e d a c t i o n p o t e n t i a l s by i n t r a c e l l u l a r c u r r e n t pu l se i n j e c t i o n s . 59 - x i i -FIGURE Page 14 I n te rna l C s + - a p p l i c a t i o n i n n e o c o r t i c a l neurons. 60 15 IPSP-at tenuat ion by i s o f l u r a n e and A l t h e s i n a f t e r K + -conductance blockade by i n t r a c e l l u l a r a p p l i c a t i o n of C s + . 62 16 Dose-dependent i n t e r f e r e n c e w i th the IPSPs i n n e o c o r t i c a l neurons w i th concomitant blockade of K + -conductances by i n t r a c e l l u l a r C s + . 63 17 E f f e c t s of i o n t o p h o r e t i c a l l y app l i ed a c e t y l c h o l i n e on n e o c o r t i c a l neurons. 71 18 Glutamate evoked responses i n neoco r t i c a l neurons. 73 19 NMDA-induced d e p o l a r i z a t i o n s i n sensor imotor l a y e r V neurons. 75 20 A h y p e r p o l a r i z i n g response to GABA app l i ed from a compound e l e c t r ode assembly. 76 21 D i f f e r e n t types of d e p o l a r i z i n g responses evoked by d e n d r i t i c a p p l i c a t i o n of GABA. 77 22 C h a r a c t e r i s t i c s of the GABA-evoked d e p o l a r i z a t i o n s i n n e o c o r t i c a l neurons. 79 23 B i c u c u l l i n e , i n h igh doses, b locked the GABA-induced d e p o l a r i z a t i o n s i n n e o c o r t i c a l neurons. 80 24 Depress ion by i s o f l u r a n e of responses to a c e t y l c h o l i n e . 82 25 Depress ion by A l t h e s i n of responses to a c e t y l c h o l i n e . 83 26 Depress ion by i s o f l u r a n e of responses to g lutamate. 84 27 Anaes the t i c depres s ion of responses to glutamate and NMDA. 85 28 P o t e n t i a t i o n of g lutamate ac t i on s and a t t enua t i on of a c e t y l c h o l i n e responses by i s o f l u r a n e . 86 29 E f f e c t s of i s o f l u r a n e a p p l i c a t i o n on equ i -amp l i tude responses t o GABA, g lutamate and a c e t y l c h o l i n e . 88 30 E f f e c t s of A l t h e s i n a p p l i c a t i o n on the a c e t y l c h o l i n e - , GABA-and glutamate-evoked responses. 89 31 Depress ion o f a c e t y l c h o l i n e , glutamate and GABA responses by high dose of i s o f l u r a n e . 90 - x i i * i -FIGURE Page 32 S e l e c t i v e anaes the t i c depress ion of a ce t y l c ho l i n e - e voked responses. 92 33 Time courses f o r anaes thet i c depress ion of ju s t -max imal responses to GABA, glutamate and a c e t y l c h o l i n e i n the presence of TTX. 94 34 Pooled data show dose-response r e l a t i o n s h i p s f o r i s o f l u r a n e -induced depress ions of d e p o l a r i z a t i o n s evoked by t r a n s m i t t e r substances. 95 35 Dose-response r e l a t i o n s h i p s f o r A l t h e s i n - i n d u c e d depress ion of d e p o l a r i z a t i o n s evoked by t r a n s m i t t e r substances. 96 36 S e l e c t i v i t y i n the i s o f l u r a n e - and A l t he s i n - e voked depress ion of neuronal respons iveness to a c e t y l c h o l i n e , glutamate and GABA. 98 37 Continuous record of i s o f l u r a n e - i n d u c e d depres s ion of the a c e t y l c h o l i n e - p o t e n t i a t i o n of g l u t ama te - a c t i on s . 99 38 A l t h e s i n - i n d u c e d depress ion of the a c e t y l c h o l i n e - p o t e n t i a t i o n of g l u t amate - a c t i on s . 100 39 Blockade of a c e t y l c h o l i n e and GABA a c t i o n s i n Mg-free pe r fu sa te . I l l 40 P o t e n t i a t i o n of NMDA responses i n the Mg-free bath ing s o l u t i o n c on t a i n i n g TTX. 112 41 Depress ion of g lutamate-responses by gradual removal of [ M g 2 + ] 0 . 115 42 Depress ion of GABA-evoked d e p o l a r i z a t i o n by low [ M g 2 + ] 0 . 116 43 Gradual p o t e n t i a t i o n of a c e t y l c h o l i n e - a c t i o n s due to a s equen t i a l i nc rease of [ M g 2 + ] 0 . 117 44 GABA ac t i on s were g r adua l l y p o t e n t i a t e d by sequent i a l i n c rea se i n [ M g 2 + ] 0 . 118 45 Dose-response curves showing the e f f e c t s of [ M g 2 + ] Q on the d e p o l a r i z a t i o n s evoked by a c e t y l c h o l i n e , g lutamate and GABA. 120 46 E f f e c t s of changing Ca- and Mg-concentrat ions on glutamate a c t i o n s . 121 47 Anae s the t i c a c t i o n s on a c e t y l c h o l i n e , g lutamate- and GABA-induced responses dur ing a p p l i c a t i o n of Mg-free media. 122 - x i v -FIGURE Page 48 I s o f l u r ane induced an a t t e n u a t i o n of the a f t e r h y p e r -p o l a r i z a t i o n s . 129 49 Depress ion of the a f t e r h y p e r p o l a r i z a t i o n s by a p p l i c a t i o n s of A l t h e s i n . 131 50 Dose-response r e l a t i o n s h i p s f o r the depres s ion of the a f t e r -hype r p o l a r i z a t i o n s produced by a p p l i c a t i o n s of i s o f l u r a n e and A l t h e s i n . 132 51 I s o f l u r ane and A l t h e s i n induced depress ions of the a f t e r h y p e r p o l a r i z a t i o n s where b i c u c u l l i n e meth iod ide had been a d d i t i o n a l l y a p p l i e d . 134 52 Changes i n r e s t i n g membrane p o t e n t i a l , i nput r e s i s t a n c e , and a f t e r h y p e r p o l a r i z a t i o n ampl itude or du r a t i on induced by i s o f l u r a n e and A l t h e s i n . 135 53 P o t e n t i a t i o n of g lutamate- induced inc reases i n i n c u l t u r e d hippocampal neurons by 0.5 MAC i s o f l u r a n e under c o n d i t i o n s favour ing Qu i s - recepto r - sub type s t i m u l a t i o n . 140 54 I s o f l u r ane (1.75 MAC) i n h i b i t i o n of i nc reases i n [ Ca 2 + ] - j produced by glutamate a p p l i c a t i o n under NMDA c o n d i t i o n s . 141 55 I s o f l u r ane (2.5 MAC) blockade of i nc rease i n [ C a 2 + ] j by g lutamate a p p l i c a t i o n under Quis c o n d i t i o n s . 142 56 Vo l tage-gated i nc rease i n [ C a 2 + ] j produced by K + bo lus i n j e c t i o n s were depressed by i s o f l u r a n e a d m i n i s t r a t i o n under Quis c o n d i t i o n s . 145 ACKNOWLEDGEMENTS I wish to thank a l l the members of the Department of Pharmacology 3 Therapeut ic s who o f f e r e d help and adv i ce . I p a r t i c u l a r l y wish to express my g r a t i t u d e to Dr. K. Le ighton f o r h i s support. I am a l so g r a t e f u l t o Dr. B. MacLeod and Dr. B. Sa s t ry f o r t h e i r help and encouragement and to Dr. K. Baimbridge f o r p rov id i ng me w i t h the f a c i l i t i e s to measure i n t r aneu rona l ca l c i um c o n c e n t r a t i o n . I would l i k e to thank Mr. L. Corey f o r h i s a s s i s t ance i n the computer a n a l y s i s of p a r t of the da ta . I a l s o app rec i a te the e f f o r t and pa t i ence o f Ms. J . Swetnam and Ms. M. Wong f o r t h e i r help i n t yp ing and p r i n t i n g the f i n a l d r a f t of the t h e s i s . La s t but not l e a s t , I wish to express my g r a t i t u d e t o Dr. E. P u i l . I would l i k e to thank him f o r a l l h i s h e l p , adv i ce , guidance and support . H i s c o n t r i b u t i o n s are g r a t e f u l l y acknowledged. - x v i -LIST OF ABBREVIATIONS A c e t y l c h o l i n e A f t e r d e p o l a r i z a t i o n A f t e r h y p e r p o l a r i z a t i o n 2-ami no-5-phosp honovalerate 4 -aminopyr id ine Y -aminobutyrate Ampere( s) A r t i f i c i a l c e r eb r o sp i n a l f l u i d C y c l i c guanosine monophosphate 2 , 4 - d i n i t r o p h e n o l E f f e c t i v e c oncen t r a t i on E f f e c t i v e dose E x c i t a t o r y po s t s ynap t i c p o t e n t i a l E x t r a c e l l u l a r ca l c i um concen t ra t i on E x t r a c e l l u l a r magnesium concen t ra t i on Glutamate I n h i b i t o r y po s t s ynap t i c p o t e n t i a l Input r e s i s t a n c e I n t r a c e l l u l a r f r e e ca l c i um concen t ra t i on I n t r a c e l l u l a r f r e e magnesium concen t ra t i on Ka inate N-methy 1-D-aspartate Minimum a l v e o l a r concen t r a t i on Minute(s ) Ohm(s) P r o t e i n k inase C Qu i squa late Rest ing membrane p o t e n t i a l R e t i c u l a r a c t i v a t i n g system Second(s) Standard d e v i a t i o n Standard e r r o r of mean Te t r ae t hyl ammoni um Te t rodo tox i n Vo l t s - X V I 1 -In the name of God, Most Grac ious , Most M e r c i f u l " Say: He i s God, the One and Only. God the E t e r n a l , Ab so lu te . He beget teth not, nor i s He begotten. And t he re i s none L i k e unto Him." ( T r a n s l a t i o n from the Holy Qur 'an) I wish to ded ica te t h i s t h e s i s to my pa ren t s , N a b i l a and Mostafa E l - B e h e i r y , who have s t rugg led cont inuous l y f o r my w e l l - b e i n g . 1 INTRODUCTION H. EL-BEHEIRY 1 1.1 General scope of t o p i c Dur ing the past two decades, v i r t u a l l y hundreds of a c t i on s of general anae s the t i c s on ve r t eb ra te and i n v e r t e b r a t e neurons have been de s c r i bed . However, the re are no hypotheses t ha t adequately e x p l a i n the neuronal mechanisms by which these agents produce the unconscious s t a t e . There are two main reasons f o r t h i s . F i r s t , anaes thet i c a c t i o n s have not been thorough ly s t ud i ed on r e l e van t c e l l types i n the c e n t r a l nervous system (CNS). Neu rophy s i o l og i ca l and b iochemica l (metabo l i c ) ev idence i n d i c a t e s t h a t n e o c o r t i c a l neurons are the l i k e l y t a r g e t s i t e s f o r anae s the t i c agents. C e r t a i n t e c h n i c a l d i f f i c u l t i e s and a modest understanding of the p h y s i o l o g i c a l mechanisms of arousal have l i m i t e d the prev ious i n v e s t i g a t i o n s t o the more r e a d i l y s tud ied neurons of s u b c o r t i c a l s t r u c t u r e s or t i s s u e c u l t u r e d neurons. Second ly , there i s an absence of a common s p e c i f i c i t y i n the chemical s t r u c t u r e of hydrocarbons, e t he r s , b a r b i t u r a t e s , p y r im id i ne s , s t e r o i d s and ino rgan ic compounds t h a t possess the anaes the t i c p rope r t y . Comprehensive r epo r t s t ha t have compared the a c t i o n s of s t r u c t u r a l l y d i s s i m i l a r anae s the t i c s on the same p h y s i o l o g i c a l type of neuron are r a r e , d e s p i t e a pe rce i ved importance of such expe r imenta t i on . In the p resent r e sea r ch , the e f f e c t s of two c h e m i c a l l y d i s t i n c t anaes-t h e t i c s , i s o f l u r a n e - - a r e l a t i v e l y r e cen t , v o l a t i l e agent, and A l t h e s i n - -a s t e r o i d a l p r e p a r a t i o n , were s tud ied on n e o c o r t i c a l neurons of the b r a i n i n order t o address f ou r fundamental que s t i on s : (a) do s t r u c t u r a l l y d i f f e r e n t anae s the t i c s have s i m i l a r (depressant) e f f e c t s on the same neuronal type? H. EL-BEHEIRY 2 (b) i s anae s the t i c - i nduced depress ion of neuronal a c t i v i t y i n neocortex due to an a t t e n u a t i o n of e x c i t a t i o n or an enhancement o f p r e v a i l i n g i n h i b i t i o n a t synapses (or both)? (c) by what neuronal mechanisms do anaes the t i c s obtund n e o c o r t i c a l arousal ? (d) what are the p l a u s i b l e i o n i c mechanisms f o r the anae s the t i c i n t e r f e r e n c e w i th e x c i t a t o r y or i n h i b i t o r y processes? A major hypothes i s evolved dur ing the course of these i n v e s t i g a t i o n s , i . e . , general anae s the t i c s act w i th vary ing degrees of e f f i c a c y on neuronal a c t i v i t i e s i n the neocortex by s e l e c t i v e l y depress ing c e r t a i n e l e c t r i c a l p roces ses . Neuronal e x c i t a b i l i t i e s are decreased in a s p e c i f i c manner and a c o r t i c a l awareness of the ex te rna l and i n t e r n a l environments are consequent ly obtunded, r e s u l t i n g i n the unconscious s t a t e . In o rder to more f u l l y understand the b a s i s f o r t h i s , a synops is of c u r r e n t ev idence as to how consc iousness i s brought about in unanaesthet ized b r a i n i s p resented below. 1.2 The Conscious System In The B r a i n 1.2.1 D e f i n i t i o n s . Among a l l aspects of human behaviour consc iousness i s probably the most d i f f i c u l t to d e f i n e . This i s p a r t l y because consc iousness i s phenomenological and i s regarded as an a t t r i b u t e o f the CNS; t h i s i s somewhat analogous to the p roper ty of " l uminescence " which i s cons ide red an a t t r i b u t e o f c u l t u r e d luminous b a c t e r i a (White 1987). Recent attempts to v i s u a l i z e consc iousness as a multicomponent system have met w i t h some success ( K i s s i n 1986). The va r ious subsystems i n vo l ved i n the processes produc ing consc iousness of the b r a i n are c l o s e l y i n t e g r a t e d i n f u n c t i o n and e l e c t r i c a l a c t i v i t y . As a consequence of the interdependency of the modular o r g a n i z a t i o n , consc iousness i s u s u a l l y exper ienced as a H. EL-BEHE IRY 3 s i n g l e , though i n t e g r a t ed e n t i t y . In the mult icompartmental c o n c e p t u a l i z a -t i o n , consciousness has o b j e c t i v e ( a l e r t ne s s ) and s u b j e c t i v e (awareness) components. Awareness i n tu rn may be vague, i . e . , general i n content o r s p e c i f i c i n d i s c r i m i n a t i o n ( s e l f - awa rene s s ) . In a d d i t i o n , consc iousness i s c h a r a c t e r i z e d by a " l e v e l of energy" t h a t has been r e f e r r e d to as " a r o u s a l " o r " a c t i v a t i o n " and q u a l i t a t i v e l y , as " a f f e c t " . Thus a beam of awareness may be focused i n one d i r e c t i o n or another under the paradigm of " a t t e n t i o n " . These components — a c t i v a t i o n , a l e r t n e s s , a r ou s a l , . a f f e c t , the dual f a ce t s of awareness and a t t e n t i o n - - represent d i f f e r e n t aspects of consc iousness . The above terms de sc r i be d i f f e r e n t dimensions of consc iousness , each of which c o n s t i t u t e s a separate and d i s c r e t e process under the c o n t r o l of a de f i ned anatomical and p h y s i o l o g i c a l system in the b r a i n . 1.2.2 I n t eg r a t i on of f u n c t i o n and a c t i v i t y of the consc ious  subsystems. As mentioned above, the va r ious subsystems of consc iousness are c l o s e l y i n t eg r a ted i n f u n c t i o n and a c t i v i t y . For example, an i nc rea se i n arousa l brought about by an inc rease i n mo t i v a t i ona l e x c i t a t i o n i s u s u a l l y a s s o c i a t e d , on an almost one-to-one bas i s w i th comparable i nc rea se s i n a c t i v a t i o n , a l e r t n e s s , awareness and a t t e n t i o n . The re fo re , mo t i v a t i ona l - emot i ona l arousa l produces an e l e c t r o p h y s i o l o g i c a l a c t i v a t i o n of the b r a i n , which i s t r a n s l a t e d epiphenomenally i n t o a l e r t n e s s and awareness. Awareness i s focused through a t t e n t i o n onto the c o g n i t i v e l y and m o t i v a t i o n a l l y s i g n i f i c a n t events i n the i n t e r n a l and ex te rna l enviroments such t h a t the f i n a l sequence of d r i v e - o r i e n t e d behav ioura l responses can be produced or repeated. 1.2.3 Func t i ona l neuroanatomy o f the consc iousness system (a) A c t i v a t i n g systems of the b r a i n . These are generated by the ascending r e t i c u l a r a c t i v a t i n g pathways t h a t p r o j e c t from the medial p o r t i o n of the H. EL-BEHEIRY 4 bra instem i n t o the do r sa l medial thalamus whereby impulses r a d i a t e to a l l po r t i on s of the basal g a n g l i a , l i m b i c system and most s i g n i f i c a n t l y , to the neocortex (Moruzzi and Magoun 1948; Mori son and Dempsey 1962). The r e t i c u l a r a c t i v a t i n g system (RAS) c o n t r o l s the 'energy l e v e l s ' i n the b r a i n . Hence, a s t a t e of d i f f u s e background a c t i v a t i o n of the ce reb ra l co r tex can be mainta ined f o r the consc ious s t a t e (Webb 1983). The 'pacemaker ' of the RAS i s the locus coeru leus of the pons. E l e c t r i c a l a c t i v i t y i n the locus coeru leus i s r e f l e c t e d by the presence of 6 - (h igh a c t i v i t y ) or y- (very high a c t i v i t y ) waves i n the c o r t i c a l l y monitored EEG (Snyder and S co t t 1972). (b) A l e r t n e s s subsystem. A l e r t n e s s i s the behav ioura l l e v e l o f consc iousness when the RAS i s m i l d l y a c t i v e as i n the s o - c a l l e d r e s t i n g , r e l axed s t a t e ( K i s s i n 1986). An i n d i v i d u a l may be consc ious but gene ra l l y una t t en t i v e dur ing which the thalamus induces a-waves i n the EEG. This i s d i f f e r e n t from the behav ioura l l e v e l of awareness which r e f l e c t s the a c t i v a t i o n of the awareness system by the RAS of the b r a i n (see below). (c) Awareness systems of the b r a i n . These c o n s i s t of general and se l f -awareness subsystems of the bra ins tem. Both are connected to the a c t i v a t i n g systems and to each o ther . The general awareness system c o n s i s t s of the p o s t e r i o r hypothalamus, the dorsa l median and a n t e r i o r n u c l e i of the thalamus, the globus p a l l i d u s and the putamen of the basal g ang l i a and the nucleus b a s a l i s of Meynert i n the sub s tan t i a innominata of the pa l eoco r t ex . The i n d i v i d u a l anatomical u n i t s of t h i s system, though ana tom i ca l l y d i s pe r s e , operate i n an i n t e g r a t e d f a sh i on to prov ide a l l the p r i m i t i v e elements of consc iousness and a t t e n t i o n a l a c t i v i t i e s . Les ions i n t h i s system can r e s u l t i n a syndrome known as a k i n e t i c mutism, i n which the re i s no r e a c t i o n to ex te rna l s t i m u l i ( G i r v i n 1975). The se l f -awareness system i s r e spons i b l e H. EL-BEHEIRY 5 f o r the awareness of the i n t e r n a l enviroment i . e . , " one ' s s e l f . The pa r t s of the b r a i n t h a t are i nvo l ved i n the phenomenology of se l f -awareness accord ing to Mesulam and Geschwind (1978) are the nucleus accumbens s ep t i of the d iencepha lon, s ub s t an t i a innominata and the p o s t e r i o r i n f e r i o r p a r i e t a l l obes . The l a t t e r r e ce i ve c h o l i n e r g i c p r o j e c t i o n s from the d i encepha l i c and p a l e o c o r t i c a l c en t r e s . Heilman and Watson (1977) and Mesulam and Geschwind (1978) have de sc r i bed a c h a r a c t e r i s t i c behaviour i n p a t i e n t s w i th u n i l a t e r a l l e s i o n s i n the i n f e r i o r p a r i e t a l lobe secondary to e i t h e r a s t r oke or tumor. The i n d i v i d u a l o f ten behaves as though one -ha l f of the wor ld d i d not e x i s t a t a l l , i . e . , a p a t i e n t may shave on ly one s i de of h i s face or another p a t i e n t may dress on ly ha l f of h i s body. (d) The a t t e n t i o n apparatus. A c t i v a t i o n of the thalamo-basal g a n g l i a r complex, bra instem general awareness system and the nucleus accumbens s e p t i , s u b s t a n t i a innominata and p o s t e r i o r i n f e r i o r lobe se l f -awareness system produces a g ene r a l i z ed s t a te of a l e r t n e s s and awareness. Such a c t i v a t i o n would be meaningless unless i t were focused on c r i t i c a l events i n the environment. The sequence of events can be r econ s t ruc ted as f o l l o w s : The a t t e n t i o n apparatus (1) assesses environmental events t ha t are deemed b i o l o g i c a l l y s i g n i f i c a n t , (2) d i r e c t s more s p e c i f i c aspects of consc iousness to these event s , (3) t i e s together the events d i r e c t l y i n t o the a c t i v a t e d s e l f - c o n c e p t system and (4) m o b i l i z e s a l l reserves to deal w i th them most e f f e c t i v e l y . The neocortex i s the s i t e of these complex c o n s c i o u s n e s s - d i r e c t i n g mechanisms ( c f . K i s s i n 1986). The sensory co r t ex and the i n f e r i o r temporal lobe r e c e i v e primary sensory (processed) i n f o rma t i on from the thalamus and cont iguous hypothalamus as we l l as the basal g a n g l i a , f o r c o g n i t i v e p roces -s ing and m o t i v a t i o n a l emot i ona l /eva l ua t i on , r e s p e c t i v e l y . In tegrated H. EL-BEHEIRY 6 i n f o rmat i on from these c o n t r o l cen t re s f lows to the p r e f r o n t a l area which i n t u r n c o n t r o l s the f l ow through the amygdaloid-hippocampal complex, i . e . , f o r the a t t e n t i o n - d i r e c t i n g a c t i v i t i e s . In a d d i t i o n , the re e x i s t s a p r e f r o n t a l -tha lamic feedback arrangement which i n h i b i t s e l e c t r i c a l a c t i v i t y i n the unattended sensory channe l s . Thus a t t e n t i o n can be concent ra ted towards a g i ven sensory moda l i t y . 1.2.4 N e o c o r t i c a l a c t i v a t i o n . A f u n c t i o n i n g consc iousness system i s c h a r a c t e r i z e d by a steady s t a te l e v e l of c o r t i c a l e x c i t a t i o n (Matsumura e t a l . 1988) which i s p r e r e q u i s i t e t o the f a s t , ' p h a s i c ' t r a n s f e r of i n f o rma t i on b i d i r e c t i o n a l l y i n the a t t e n t i o n system. N e o c o r t i c a l a c t i v a t i o n r e s u l t s from s u b c o r t i c a l as we l l as i n t r a c o r t i c a l ( h o r i z o n t a l or v e r t i c a l ) e x c i t a t o r y t r an sm i s s i on (Enzure and Oshima 1981a; G i u f f r i d a and Ru s t i o n i 1989). C h o l i n e r g i c ascending r e t i c u l a r pathways p r o j e c t to the deep c o r t i c a l l a y e r s (Pauly et a l 1989) o r i g i n a t i n g from the m idb ra i n , pons, thalamus and magnoce l l u l a r neurons i n the basal f o r e b r a i n (Shute and Lewis 1967; Divac 1975; Hoover and Jacobowitz 1979; Lehmann e t a l . 1980; Kimura e t a l . 1981; Spencer et a l 1986). Adrenerg ic and h i s t am ine r g i c i n f l u e n c e s i n n e o c o r t i c a l a c t i v a t i o n may be s i g n i f i c a n t ( R o t h b a l l e r 1956; L ing and Fou lk s 1959; Wanatabe et a l . 1984; Re iner and McGeer 1987) as i n d i c a t e d by demonstrat ions of s p e c i a l i z e d groups of b iogen ic amine-conta in ing neurons i n the midbra in and pons t h a t p r o j e c t d i f f u s e l y to the co r tex (Reader et a l . 1988). Adren-e r g i c and se ro tonerg i c ascending systems to the neocortex have been de sc r i bed ( L e v i t t and Moore 1978; L idov et a l . 1980) a l though these are more r e l a t e d t o the s leep-waking c y c l e (Aston-Jones and Bloom 1981) than to the maintenance of consc iousness . More r e c e n t l y , d i r e c t morpho log ica l ev idence has been i n t e r p r e t e d f o r the coex i s t ence of the neuropeptide substance P w i t h a c e t y l c h o l i n e i n RAS neurons (V incent e t a l . 1983). H. EL-BEHEIRY 7 1.2.5 A c t i v a t i n g neu ro t ran sm i t te r s i n the neocortex. A c e t y l c h o l i n e (ACh) i s cons idered a major c o r t i c a l ' a c t i v a t i n g t r a n s m i t t e r ' because n e o c o r t i c a l a c t i v a t i o n can be e l i c i t e d with cho l i nomimet i c s and b locked by a t r op i ne (Funderburk and Case 1951). A r o l e f o r ACh at n e o c o r t i c a l synapses has been suggested i n the generat ion of a s t a te of enhanced respons iveness of neurons. A p p l i c a t i o n s of ACh enhance the neuronal f i r i n g evoked by e l e c t r i c a l s t i m u l a t i o n of s ynap t i c inputs ( l i k e l y g lu tamaterg i c pathways) and prolongs a f t e r d i s c h a r g e s such tha t the sp ike bu r s t s are more numerous and prolonged i n du r a t i on ( K r n j e v i c 1987, 1988). A l s o , exogenous ACh can induce r e p e t i t i v e f i r i n g which i s be l i e ved to be p r e r e q u i s i t e to the generat ion of mnemonic t r a c e s ( K r n j e v i c e t a l . 1971a). Another l i k e l y major t r a n s m i t t e r i n the neocortex i s g lutamate (Glu) ( K r n j e v i c and Phi 11 i s 1963a; McLennan 1983; P u i l and Benjamin 1988). In c o n t r a s t t o ACh, Glu has a r a p i d on se t /o f f s e t a c t i o n . G l u , a c t i n g as a t r a n s m i t t e r , may be r e spon s i b l e f o r the f a s t t r a n s f e r as we l l as p roces s ing of i n f o rma t i on at synapses i n pathways f o r consc ious p roces s ing ( K r n j e v i c 1987). However, ascending c h o l i n e r g i c e x c i t a t i o n of the neocortex modulates the r a t e of t r a n s f e r of i n f o r m a t i o n , i n t r a c o r t i c a l l y , as w e l l as between c o r t i c a l and s u b c o r t i c a l s t r u c t u r e s (Conti e t a l . 1987; G i u f f r i d a and R u s t i o n i 1989). For example, i n a comatose p a t i e n t , the t o n i c ACh e x c i t a t i o n may be a t tenuated to a lower l e v e l than t h a t present i n an a l e r t i n d i v i d u a l . Hence, the p roces s ing of sensory i n f o rmat i on i s obtunded i n the unconscious s t a t e . A c o n t r i b u t i o n of i n h i b i t o r y t r an sm i s s i on to n e o c o r t i c a l a c t i v a t i o n has y e t to be e l u c i d a t e d . However, some repor t s suggest a model of neuronal c i r c u i t r y t ha t may make up the elementary s t r u c t u r e of c o r t i c a l a c t i v a t i o n (Enzure and Oshima 1981b). In a d d i t i o n to e x c i t a t o r y neurons, i n h i b i t o r y H. EL-BEHEIRY 8 GABAergic neurons have been po s tu l a ted i n the model; these have been i d e n t i f i e d dur ing s t i m u l a t i o n of the RAS i n awake c a t s ( Inubushi e t a l . 1978a, b; Enzure and Oshima 1981a). Consequently, GABA cou ld be an important t r a n s m i t t e r r e spons i b l e f o r a negat ive feed-back loop i n the neuronal o r g a n i z a t i o n of the consciousness system. 1.2.6 The ' a c t i v a t i n g t r a n s m i t t e r s ' and t h e i r i o n i c mechanisms of a c t i o n 1.2.6.1 A c e t y l c h o l i n e (ACh). E x c i t a t i o n mediated by ACh or by other t r a n s m i t t e r s i s brought about through the f o l l o w i n g i o n i c membrane mechanisms: (a) a decrease i n the r e s t i n g K + conductance, (b) a s lowing of r e p o l a r i z a t i o n due t o . reduct ion i n the de layed K + r e c t i f y i n g conductance, (c) a r educ t i on i n the slow a f t e r h y p e r p o l a r i z a t i o n (AHP) due to +2 + i n h i b i t i o n of the slow Ca - a c t i v a t e d K conductance and (d) i n h i b i t i o n of the M-current . (a) Decrease i n the r e s t i n g K + conductance. K rn jev i c " and h i s a s s o c i -ates (1971a) f i r s t demonstrated t h i s mechanism in n e o c o r t i c a l neurons of the l i g h t l y anaes the t i zed c a t i n i n v i vo exper iments. Subsequent i n v e s t i g a t i o n s by Woody et a l . (1978) conf i rmed these unconvent ional a c t i o n s of ACh i n con s c i ou s l y f r e e l y moving c a t s . McCormick and P r i n c e (1986) a l s o demon-s t r a t e d the a c t i o n s of ACh on neurons i n i n v i t r o s l i c e s of a n t e r i o r c i n g u l a t e co r t ex of guinea p i g s . In the e a r l y s t ud i e s a tendency f o r the input r e s i s t a n c e to i nc rea se dur ing an a p p l i c a t i o n of ACh and a l s o a r e v e r s i b l e p o t e n t i a l c l o s e to -100 mV f o r the d e p o l a r i z i n g a c t i o n s were observed. S ince the re was no conv inc ing evidence of inward (anomalous) r e c t i f i c a t i o n i n these neurons, the r e s u l t s cou ld be best exp l a i ned by a r educ t i on i n C I " or K + conductance (both ions have an e q u i l i b r i u m p o t e n t i a l c l o s e t o -100 mV). I n t r a c e l l u l a r i n j e c t i o n s o f C I " which produced l a r ge p o s i t i v e s h i f t s i n the C I " e q u i l i b r i u m p o t e n t i a l and the H. EL-BE HE IRY 9 IPSP r eve r s a l p o t e n t i a l d i d not obv ious l y change the c h a r a c t e r of the neuronal responses t o ACh. There fo re , K r n j e v i c e t a l . (1971a) concluded t h a t ACh probably acted by reducing membrane conductance f o r K + . As 2+ support ing ev idence, Ba , which i s known to i n t e r f e r e r a t he r s p e c i f i c a l l y w i t h movements of K + i n a v a r i e t y of e x c i t a b l e t i s s u e s , was shown to e x c i t e c o r t i c a l c e l l s i n a manner s i m i l a r to ACh ( K r n j e v i c e t a l . 1971b). (b) Reduct ion i n the delayed K* r e c t i f i e r . This e f f e c t ( K r n j e v i c 1971a; Woody et a l . 1978; Bernardo and P r i n c e 1982), which i s mimicked by i o n t o p h o r e t i c a p p l i c a t i o n of Ba^ + ( K r n j e v i c e t a l . 1971b) leads to a " s l ow ing down" of r e p o l a r i z a t i o n , i . e . , the f a l l i n g phase of the a c t i o n p o t e n t i a l s , and enhances r e p e t i t i v e f i r i n g and a f t e r d i s c h a r g e s . 2+ + (c) Depress ion of the Ca a c t i v a t e d K conductance. The depress ion of t h i s net outward c u r r e n t abo l i s he s the AHP. McCormick and P r i nce (1986) showed t h a t when ACh was app l i ed by pressure e j e c t i o n , the po s t sp i ke AHP i n n e o c o r t i c a l neurons was s u b s t a n t i a l l y reduced and even rep laced by an a f t e r d e p o l a r i z a t i o n (ADP). Th i s f i n d i n g i s i n agreement w i t h the e a r l y obse rva t ions ( K r n j e v i c e t a l . 1971a, 1978a,b). However, the exact mechanism of the AHP depres s ion i s not we l l understood; the e f f e c t cou ld be mediated 2+ 2+ by an i nc reased Ca b ind ing t o ca lmodu l i n (a Ca - b i nd i ng p r o t e i n ) , a 2+ r educ t i on i n Ca - i n f l u x , d imin i shed i n t e r n a l C a - s eque s t r a t i o n or i t cou ld be due to a more d i r e c t i n t e r a c t i o n between the a c t i v a t e d ACh-receptor and the r e l e v a n t K- ionophores ( c f . K r n j e v i c 1984). On the o ther hand, the replacement of the AHP by an ADP may be due to an unmasking of a slow 2+ + d e p o l a r i z i n g event mediated by Ca or Na which o r d i n a r i l y would be obscured or b locked by the AHP (McCormick and P r i n c e 1986). H. EL-BEHEIRY 10 (d) Suppress ion of the M-cur rent . There i s now good reason to b e l i e v e t h a t the muscar in ic a c t i on s lead to s e l e c t i v e suppress ion of a v o l t a g e - and t ime-dependent, n o n - i n a c t i v a t i n g K -cu r ren t generated by r e l a t i v e l y smal l d e p o l a r i z a t i o n s . As a consequence of the e x c i t a t i o n of t h i s c u r r e n t , the neuron tends to h ype rpo l a r i ze and r e p e t i t i v e f i r i n g subs ides . Suppress ion of t h i s slow outward cu r r en t g r e a t l y f a c i l i t a t e s the f i r i n g and a f t e r d i s -charges induced by concomitant a c t i o n s of e x c i t a t o r y t r a n s m i t t e r s l i k e G l u . The M-current was f i r s t de sc r ibed i n b u l l f r o g sympathetic g ang l i a (Brown and Adams 1980), then i n hippocampus ( H a l l i w e l l and Adams 1982; Malenka e t a l . 1986; Moore e t a l . 1988), f o l l owed by guinea p i g o l f a c t o r y co r tex (Constant i and Sim 1987) and human neocortex ( H a l l i w e l l 1986). Because Gclhwiler and D re i f u s s (1982) repor ted t ha t ACh was i n e f f e c t i v e i n the absence 2+ of Ca i n hippocampal neurons, a t l e a s t par t of the M-current may be a r e s u l t of a Ca-dependent K-conductance as specu lated e a r l i e r by K r n j e v i c (1977). In more recent vo l tage-c lamp i n v e s t i g a t i o n s of r a t sympathetic g a n g l i a , B e l l u z z i e t a l . (1985) found t h a t muscarine b locks on ly inward-Ca 2+ cu r r en t s and i s i n e f f e c t i v e i n the absence of e x t r a c e l l u l a r Ca . Hence, they concluded tha t the "M-channels " are r e a l l y C a - a c t i v a t e d K-channels and t h a t t h e i r apparent v o l t a g e - and t ime-dependencies r e f l e c t the membrane p r o p e r t i e s of the Ca-channel s . The idea tha t ACh ac t s p r i m a r i l y on Ca-channels i s a t t r a c t i v e because i t p rov ides a u n i t a r y exp l ana t i on f o r a l l musca r in i c a c t i o n s d i scus sed thus f a r f o r CNS neurons. C h o l i n e r g i c i n h i b i t i o n has been repor ted f o r a l l l e v e l s of the nervous system, i n c l u d i n g the ce reb r a l c o r t e x , neo s t r i a tum, thalamus, hypothalamus, b ra in s tem, s p i na l cord and sympathet ic and parasympathet ic g ang l i a (K rn jev i c * 1974a; L i b e t 1970; Z ieg lgansberger and R e i t e r 1974; H a r t z e l l e t a l . 1977; Dodd e t a l . 1981; Bernardo and P r i n ce 1982; f f r e n c h - M u l l e n e t a l . H. EL-BEHEIRY 11 1983). I n h i b i t i o n has been observed j u s t p r i o r to the d e p o l a r i z i n g ACh response, i n many cases . C h o l i n e r g i c i n h i b i t i o n had been observed most ly w i t h i n the s u p e r f i c i a l l a ye r s (I I and I I I ) of the ce reb ra l co r tex ( K r n j e v i c 1974a). I t i s a l so present i n l a y e r V, a l though the i n h i b i t o r y responses i n neurons i n the s u p e r f i c i a l l a y e r are con s ide rab l y l a r g e r (McCormick and P r i n c e 1986). In the neocortex, c h o l i n e r g i c i n h i b i t i o n may be a consequence of GABA-ergic in terneurons a c t i v a t e d by the e x t r a c e l l u l a r l y a p p l i e d ACh, which impinge on the impaled neuron thereby producing IPSPs (Haas 1982; McCormick and P r i nce 1986). The slowness i n ACh a c t i o n on c o r t i c a l neurons suggests an involvement of a secondary messenger system in the t r an sduc t i on of the response. C y c l i c GMP (cGMP) does not mimick the muscar in i c a c t i on s of ACh i n s u b c o r t i c a l neurons ( K r n j e v i c e t a l . 1976; Bernardo and P r i n ce 1982). However, the a c t i o n s of ACh cou ld be mediated i n t r a c e l l u l a r ^ by cGMP and by a cGMP-dependent p r o t e i n k inase i n n e o c o r t i c a l neurons (Woody and Gruen 1988). 1.2.6.2 S-Glutamate ( G l u ) . The e x c i t a n t e f f e c t of Glu i s a r e s u l t of the d e p o l a r i z a t i o n of the neuron to the t h r e s ho l d f o r vo l tage-dependent sp i ke a c t i v a t i o n . An involvement of another vo l tage-dependent process a s s o c i a t ed w i th the r e cep to r - coup l ed ionophore complex c o n t r i b u t e s to the conductance change measured dur ing glutamate e x c i t a t i o n a f t e r the blockade of sp ike genes i s . The net change represents a combinat ion of both voltage-dependent and vo l tage- independent behaviour of the i o n i c channels a c t i v a t e d by the r e s p e c t i v e i n t e r a c t i o n s of Glu at presumed recepto r s f o r N-methy l -D-aspartate (NMDA) and a t non-NMDA recepto r s f o r qu i s qua l a t e (Quis) and ka inate ( ka ) . The d i f f e r e n t i a t i o n between NMDA and non-NMDA responses has been made most ly on the ba s i s of b lockade a t NMDA recepto r s by s p e c i f i c an tagon i s t s such as 2-amino-5-phosphonovalerate (2-APV) o r r e c e n t l y H. EL-BEHEIRY 12 MK-801 and by Mg 2 + (Hwa and A v o l i 1984; Nowak e t a l . 1984; Thomson 1986; Coan e t a l . 1987; Mayer and Westbrook 1987; P u l l and Benjamin 1988; Col l i n g r i dge and L e s t e r 1989). The v o l t a g e - s e n s i t i v i t y of the c a t i o n i c channels a c t i v a t e d by G l u - i n t e r -a c t i on s at NMDA recep to r s i s a d i r e c t consequence of a vo ltage-dependent 2+ ?+ blockade by Mg (Nowak e t a l . 1984). This blockade occurs when Mg ente r s the a c t i v a t e d channels and decreases i n magnitude on d e p o l a r i z a t i o n , thereby c o n f e r r i n g a b i s t a b l e s t a te of the membrane p o t e n t i a l (MacDonald e t a l . 1982; Thomson 1986). NMDA induces an inward ly r e c t i f y i n g cu r r en t and a b i s t a b l e s t a te change in the input/output behaviour of the neuron; in n e o c o r t i c a l neurons t h i s can lead to a g rea te r tendency to f i r e a c t i o n p o t e n t i a l s r e p e t i t i v e l y and to develop pacemaker - l i ke o s c i l l a t i o n s in membrane p o t e n t i a l s (Flatman et a l . 1986). The d e p o l a r i z a t i o n s evoked by NMDA and non-NMDA agon i s t s have reve r sa l p o t e n t i a l s near zero m i l l i v o l t s . The r eve r s a l p o t e n t i a l s become more hype rpo l a r i zed w i th p rog re s s i ve removal of e x t r a c e l l u l a r N a + ; t h i s suggests t h a t both types of agon i s t s i nc rease membrane p e r m e a b i l i t y to Na + + and K . The r e v e r s a l p o t e n t i a l of non-NMDA responses i s much more s e n s i t i v e to changes i n ex te rna l [Na + ] whereas the r e ve r s a l p o t e n t i a l f o r the NMDA responses i s markedly a f f e c t e d by changing the ex te rna l [Ca ] ( c f . Padjen and Smith 1981; P u i l 1981). In c u l t u r e d s p i n a l cord neurons, a p p l i c a t i o n s of Glu t h a t a c t i v a t e recepto r s of the NMDA-subtype i nc rea se the 2+ 2 + i n t r a c e l l u l a r Ca - a c t i v i t y measured w i th Ca - s e n s i t i v e f l u o r e s c e n t 2+ probes. Here, Mg can be used to d i s t i n g u i s h the NMDA-receptor-coupled 2+ 2+ Ca - i n f l u x from the voltage-dependent source of Ca - i n f l u x t h a t a l so i s i n i t i a t e d by the NMDA-actions (MacDermott e t a l . 1986). In neocortex, Glu a p p l i c a t i o n produces t e t r o d o t o x i n ( T T X ) - i n s e n s i t i v e decreases i n H. EL-BEHEIRY 13 e x t r a c e l l u l a r [Na ] , [Ca ] and [Mg ] measured w i t h s e l e c t i v e i o n - s e n s i t i v e m i c roe l ec t r ode s (Pumain e t a l . 1988). Glu and the s p e c i f i c agon i s t s — NMDA, Quis and ka - - a l so produce responses t ha t are dependent on e x t e r na l [ K + ] i n d i c a t i n g t ha t the a c t i v a t e d channels i n c l ude those that are permeable to K + and t ha t the e f f l u x of K + may be p a r t l y pas s i ve or due to a c t i v e e x t r u s i o n by a pumping process (B i ih r le and Sonnhof 1983; Padjen and Smith 1983; Pumain and Heinemann 1985; Pumain et a l . 1987). 1.3 The Anae s the t i c S ta te 1.3.1 H i s t o r i c a l i n t r o d u c t i o n and d e f i n i t i o n s . Anaes thes ia i s cons ide red an American c o n t r i b u t i o n s ince the f i r s t succes s fu l p u b l i c demonstrat ion by W.T.G. Morton took p lace i n Massachussetts General Ho sp i t a l i n 1846 (B igelow 1846). However, anyone who has examined the h i s t o r y w i l l r e a l i z e t h a t i nnova t i on of such s i g n i f i c a n c e c ou l d have hard ly a r i s e n spontaneously. For example, d i e t h y l e t h e r which was the agent used by Morton, had been d i s covered i n Sw i t ze r l and around 1540 by V a l e r i u s Cordus and named Aether by F roben ius . I t s pharmacolog ica l and p a r t i c u l a r l y anae s the t i c p r o p e r t i e s i n animals were de sc r i bed by a p h y s i c i a n and a l chemi s t named Parace l sus ( c f . Smith 1986). The word " anae s the s i a " was f i r s t used as i n i t s modern sense i n the f i r s t century AD by the Greek ph i l o sophe r , D i o s c o r i d e s , who de sc r i bed the n a r c o t i c e f f e c t s of the p l an t Mandragora. I t s usage i n the E n g l i s h language i s u s u a l l y a s c r i b e d t o O l i v e r Wendell Holmes, though i t was de f i ned con s i de r ab l y e a r l i e r i n 1751 i n B a i l e y ' s E n g l i s h D i c t i o n a r y as " a de fec t of s en sa t i on " (Beecher 1968). In the c l i n i c a l c o n t e x t , Rees and Gray (1950) de f i ned anaes thes ia as a s t a t e c h a r a c t e r i z e d by n a r co s i s , ana l ge s i a and r e l a x a t i o n . Gray i n 1960 renamed t h i s t r i a d e as n a r co s i s , r e f l e x suppress ion and r e l a x a t i o n . More H. EL-BEHEIRY 14 r e c e n t l y , White (1987) attempted to de f i ne general anaesthes ia as a s t a t e i n which the CNS i s r e v e r s i b l y depressed to such a degree tha t consc iousness i s l o s t and t h a t on recovery , nothing i s r e c a l l e d r e l a t i n g to the pe r i od o f anae s the s i a . A tk in son and a s s oc i a te s (1987) prov ided a mechan i s t ic ( i . e . , o p e r a t i o n a l ) d e f i n i t i o n by suggest ing t h a t anaes thes ia i s a process of m o d i f i c a t i o n of the normal p h y s i o l o g i c a l r e f l e x response to s t i m u l i prov ided by surgery . A c c o r d i n g l y , they cons idered the t r i a d e of anaesthes ia as i n h i b i t i o n of the a f f e r e n t par t of the r e f l e x system, depress ion of the c e n t r a l s ynapt i c mechanisms of c o o r d i n a t i o n and blockade of the e f f e r e n t pa r t of the r e f l e x a r c . Th i s d e f i n i t i o n , though uncommon, prov ides the a n a e s t h e s i o l o g i s t with two main advantages. F i r s t , i t avoids the d i f f e r e n t problems i n d e f i n i n g unconsciousness (or " n a r c o s i s " ) . Secondly, i t p rov ides an o b j e c t i v e d e f i n i t i o n a p p l i c a b l e to humans as we l l as exper imental an ima l s . 1.3.2 S i t e of a c t i o n of anae s the t i c s i n the consc iousness system. Himwich (1951) was the f i r s t to suggest the concept of h o r i z o n t a l l e v e l s , w i t h i n the b r a i n t h a t were a f f e c t e d r e v e r s i b l y and s e q u e n t i a l l y by i n c r e a s i n g depths of anae s the s i a . The c e r e b r a l co r tex i s depressed before the s u b c o r t i c a l d iencepha lon, and then as anaes thes ia deepens, the mesencephalon and pons are g r a d u a l l y i n h i b i t e d . F i n a l l y the deepest anaes thes i a occurs when the v i t a l c en t re s of the medul la are depressed. More recent developments i n neurophys io logy have given support f o r a v e r t i c a l s t r a t i f i c a t i o n of the b r a i n . L i v i n g s t one (1954) v i s u a l i z e d the CNS as composed of th ree h i gh l y i n t e r r e l a t e d , though v e r t i c a l systems - - the s p e c i f i c sensory system, the non spec i f i c sensory and the motor system. The n o n s p e c i f i c sensory system has the r e s p o n s i b i l i t y of i n t e r r e l a t i n g and modulat ing a l l sensor imotor i n t e r r e l a t i o n s . Th i s n on spec i f i c system i s l o c a t e d e s s e n t i a l l y w i t h i n the m i d l i n e s t r u c t u r e s , extending from the H. EL-BEHEIRY 15 medul la to the d iencepha lon, and c o n t a i n i n g the ascending RAS. Because the r e t i c u l a r format ion has been demonstrated to be a v i t a l component f o r the c o n t r o l of consc iousness , severa l i n v e s t i g a t o r s have attempted to r e l a t e anaes thes ia to a l t e r a t i o n s i n the f u n c t i o n of the RAS (French et a l . 1953; A r d u i n i and A rdu in i 1954; K ing 1956). Two l i n e s of exper imental ev idence have a l t e r e d t h i s concept. Con-sc i ou snes s , as r e f l e c t e d i n a desynchronized EEG, i s p o s s i b l e i n c h r on i c p repa ra t i on s of exper imenta l animals w i thout the midbra in r e t i c u l a r fo rmat ion (Genovesi e t a l . 1956). A l s o , changes can be noted wi th at l e a s t some a n a e s t h e t i c s , i n the pa t t e rn of c o r t i c a l a t i v i t y , before there are changes i n the a c t i v i t y of the r e t i c u l a r fo rmat ion neurons (Winters e t a l . 1967, 1969; Darb in jan et a l . 1971; Mori e t a l . 1971, 1972; Mori 1972). Sh imoj i and B i c k f o r d (1971) observed t h a t anae s the t i c s such as halothane produced e x c i t a t o r y e f f e c t s a t the un i t a r y l e v e l i n the r e t i c u l a r core r e s u l t i n g i n an i nc reased f i r i n g of mesencephal ic r e t i c u l a r neurons. Moreover, l o c a l c e r e b r a l g lucose u t i l i z a t i o n was found to be s p e c i f i c a l l y and c o n s i s t e n t l y depressed i n neocortex and not i n bra instem of r a t s anae s the t i zed w i th pen t oba rb i t a l or halothane (Savaki e t a l . 1983; Sakabe et a l . 1985; Peschanski e t a l . 1986). From the above d i s c u s s i o n , i t i s apparent t ha t any hypothes i s which s imply proposes bra instem depress ion as the so le s i t e at which anae s t he t i c s produce unconsciousness needs c a r e f u l assessment. In summary, data on l o c a l c e r e b r a l g lucose consumption as we l l as neu rophy s i o l og i ca l obse rva t ions suggest t h a t the neocortex i s the l i k e l y s i t e of a c t i o n of a nae s t he t i c s . The exact reasons f o r such s p e c i f i c i t y are not e x a c t l y known. The r o l e of the neocortex i n the n e u r o l o g i c a l mechanisms of the anae s the t i c s t a t e r equ i r e s comprehensive i n v e s t i g a t i o n . H. EL-BEHEIRY 16 1.3.3 Mechanisms o f a c t i o n of general a n a e s t h e t i c s . Though few i n v e s t i g a t o r s would debate the importance of the neocortex as a s i t e of a c t i o n of anae s the t i c s (Robson 1967; Mori 1972; Mor i e t a l . 1971, 1972; K r n j e v i c 1974; Peschansk i e t a l . 1986), the exact t a r g e t s i t e s a t the c e l l u l a r and mo lecu la r l e v e l s remain a matter of c on s i de rab l e cont rover sy (R ichards 1980; Franks and L i eb 1982; Dluzewski e t a l . 1983; Haydon e t a l . 1984; M i l l e r 1985; Bangham and H i l l 1986). A t the mo lecu l a r l e v e l , suggest ions about the e f f e c t s of anaes thet i c molecu les on neurons have ranged w ide ly from a very general depress ion of neuronal e x c i t a b i l i t y by a uniform phys iochemica l change i n membrane p r o p e r t i e s to h i g h l y s p e c i f i c mechanisms such as p re s ynap t i c reduct ion of t r a n s m i t t e r r e l ea se or a s e l e c t i v e p ro l onga t i on of i n h i b i t o r y a c t i on s on the po s t s ynap t i c membrane ( K rn j e v i c 1972). 1.3.3.1 N o n - s p e c i f i c ( l i p i d ) hypotheses. These imply tha t anae s the t i c s ac t by changing the phy s i ca l p r o p e r t i e s of the macromolecular s t r u c t u r e of the plasma membrane, i . e . , no s p e c i f i c s i t e or r ecep to r i s r e q u i r e d . Here, the c l a s s i c a l view i s t h a t anae s t he t i c s ac t through d i s s o l u t i o n i n membrane l i p i d s as viewed by Overton (1901) and Meyer  (1899). The i r ob se rva t i on s were the e x c e l l e n t c o r r e l a t i o n between the potency of a v a r i e t y of anae s the t i c s and t h e i r l i p i d s o l u b i l i t i e s ( o l i v e / o i l p a r t i t i o n c o e f f i c i e n t ) . However, t h i s c o r r e l a t i o n does not e x p l a i n how the drugs produce anaes the s i a . The e a r l i e s t attempt to overcome t h i s d i f f i c u l t y was the c r i t i c a l volume  hypothes is of M u l l i n s (1954) which po s tu l a ted t h a t anaes thes i a occurs when a c r i t i c a l volume f r a c t i o n of anaes the t i c molecules i s ach ieved i n neuronal membranes. M u l l i n s suggested t h a t the excess volume would cause l a t e r a l pressure on the i o n i c pores of the membrane which tends t o occ lude them. As H. EL-BE HE IRY 17 a r e s u l t , impulse conduct ion and synapt i c t r an sm i s s i on would be impa i r ed . A f t e r f u r t h e r m o d i f i c a t i o n s by Paton and Pertwee (1972), Seeman (1972, 1975), Seeman and Roth (1972), Haydon et a l . (1977) and Halsey e t a l . (1978) the c r i t i c a l volume hypothes i s prov ided a reasonable model f o r e x p l a i n i n g the phenomenon of r e ve r s a l of anaes thes ia at high pressures ( M i l l e r e t a l . 1973). I t s weakness was the i m p l i c i t assumption t ha t a l l l i p i d s o l u b l e substances ( e . g . , soaps) should be anae s the t i c s and t h a t they should a l l work i n the same way. C e r t a i n compounds which are very s i m i l a r i n chemical s t r u c t u r e may have qu i t e d i f f e r e n t e f f e c t s ; f o r example, hexa f l uo ro i sop ropy lmethy l e the r i s an a n a e s t h e t i c , whereas h e x a f l u o r o d i e t h y l e t h e r i s a powerful convu l sant (R ichards 1980). The pa t t e r n of uptake and d i s t r i b u t i o n of these compounds i n the CNS a l s o should be near l y i d e n t i c a l as both processes are determined on ly by the p a r t i t i o n c o e f f i c i e n t and the l i p i d s o l u b i l i t y . The d i f f i c u l t i e s become f u r t h e r compounded when b a r b i t u r a t e s and s t e r o i d s are con s i de red , s ince both of these groups show pronounced s t r u c t u r e / a c t i v i t y r e l a t i o n s which cannot be p r e d i c t e d from the s imple c r i t i c a l volume hypo-t h e s i s (Halsey 1974). In view of these d i f f i c u l t i e s , a t t e n t i o n was d i r e c t e d towards mechanisms by which anae s the t i c s cou ld d i s r u p t the s t r u c t u r e of the l i p i d b i l a y e r to cause i n d i r e c t changes i n the a c t i v i t y of f u n c t i o n a l membrane p r o t e i n s . Three models have been proposed: (a) The p h a s e - t r a n s i t i o n hypothes i s suggests tha t l i p i d s immediately surrounding an e x c i t a b l e membrane channel are e x c l u s i v e l y i n a gel phase, which tends to ma in ta i n the patency of the channe l . The l i p i d s become f l u i d on a d d i t i o n of an anae s the t i c ( i . e . , t he re i s a p h a s e - t r a n s i t i o n ) , which a l lows the channel to c l o s e (Lee 1976). Th i s hypothes i s i s H. EL-BEHEIRY 18 supported by the f i n d i n g t h a t anaes thet i c s decrease the temperature a t which p h a s e - t r a n s i t i o n occurs i n p u r i f i e d pho spho l i p i d model membranes (Lee 1976; H i l l 1978). In a d d i t i o n , high pressure antagon izes both the development of anaes thes ia (pressure r e v e r s a l ) , and the anae s t he t i c -induced decrease i n p h a s e - t r a n s i t i o n temperature (Kamaya e t a l . 1979). (b) The l a t e r a l phase sepa ra t i on hypothes i s p o s t u l a t e s tha t under normal c o n d i t i o n s membrane phospho l i p id s c o - e x i s t i n both f l u i d and gel forms ( T r ude l l 1977). Convers ion of one form to another permits the membrane to expand or c o n t r a c t w i th le s s expenditure of energy than would be requ i red i f the membrane were pure ly f l u i d or g e l . An analogy i s p rov ided by the r educ t i on i n volume t h a t r e s u l t s when i ce me l t s . Less energy i s r equ i red to decrease the volume than to compress i c e or water w i thout a phase change. Anaes thet i c s may " m e l t " the gel phase and thereby increase the energy requ i red to d i s p l a c e p o r t i o n s of the membrane and decrease the membrane l a t e r a l c o m p r e s s i b i l i t y . Th is decrease may prevent the opening of p r o t e i n channels t h a t permit s t r a n s l o c a t i o n of ions across the po s t s ynapt i c membrane or conformat iona l changes in p r o t e i n s r e spon s i b l e f o r t r a n s m i t t e r r e l e a s e . Indeed, general anaesthe-t i c s d i s t r u p t l a t e r a l - p h a s e separat ions i n model membranes composed of two types of pho spho l i p i d s (T rude l l et a l . 1975). These e f f e c t s are p a r t i a l l y rever sed by high pressures ( T rude l l e t a l . 1975). c) The f l u i d i z e d - 1 i p i d hypothes i s s t a t e s t ha t anae s t he t i c s i nc rease the l a t e r a l and r o t a t i o n a l motions of membrane components. As i n the p h a s e - t r a n s i t i o n hypothes i s , t h i s inc rease i n the membrane d i s o rde r i s thought to f a c i l i t a t e i on i c - channe l c l o s u r e by reduc ing the s t a b i l i t y of the channel p r o t e i n s . The enhancement of the decay of the m in i a tu re H. EL-BEHEIRY 19 po s t s ynap t i c c u r r en t by i n h a l a t i o n a l agents i s c o n s i s t e n t w i th the f l u i d i z e d - 1 i p i d or p h a s e - t r a n s i t i o n hypothes i s (Gage and Hami l l 1975). The hypotheses r e l a t e d to phase t r a n s i t i o n , l a t e r a l phase separa t i on and f l u i d i z e d l i p i d suggest t h a t anae s the t i c s a c t by i n c r ea s i n g membrane f l u i d i t y . These s u f f e r from some ser ious c o n s t r a i n t s and i n c o n s i s t e n c i e s , such as the f o l l o w i n g : (1) f l u i d i t y changes i n l i p i d b i l a y e r s cannot be detected unless the anae s t he t i c concent ra t i on s which are used are we l l in excess of those used c l i n i c a l l y (Boggs e t a l . 1976; Franks and L i eb 1978; Turner and O l d f i e l d 1979; L i eb e t a l . 1982); (2) an inc rease i n temperature ( l e s s than 1°C) a l s o i nc reases membrane f l u i d i t y (Ha r r i s and Groh 1985) and t h e r e f o r e should enhance anaes the s i a . However, an i nc rease i n body temperature c o n s i s t e n t l y decreases anaes-t h e t i c potency (Eger et a l 1965) and does not produce anaes the s i a ; (3) on the other hand, c oo l i n g should o f f s e t the anaes thet i c a c t i o n because i t should oppose the f l u i d i z i n g e f f e c t accord ing to the above-mentioned models. In c o n t r a s t , R ichards and h i s a s s o c i a t e s (1978) demonstrated t h a t c o o l i n g of a nerve d i d not render i t l e s s s u s c e p t i b l e to b lockade by anae s the t i c agents ; (4) the c i s and t ran s isomers of 9 , 1 0 - t e t r a d e c a n o l , which have i d e n t i c a l a nae s t he t i c potenc ie s do not induce s i m i l a r changes in membrane f l u i d i t y ( P r i n g l e and M i l l e r 1978). One isomer lowers and the other e l e va t e s the phase t r a n s i t i o n temperature of a pho spho l i p i d membrane; (5) some chemical congeners or i somers, d i f f e r i n g on ly t r i v i a l l y from an anae s the t i c member i n chemical and phy s i c a l p r o p e r t i e s such as l i p i d s o l u b i l i t y , show convu l sant a c t i v i t y . Examples of mo lecu la r m o d i f i c a -H. EL-BEHEIRY 20 t ions that convert an anaesthetic to a convulsant molecule include addit ion of a methyl group to the a l i pha t i c side chain of barbiturates, addit ion of a single double bond in the stero id nucleus of alphaxalone and replacement of one f luor ine atom in halothane by a hydrogen atom (c f . Label la 1981, 1982). 1.3.3.2 Spec i f i c (protein) hypotheses. Because the actions of anaesthetics on membrane l i p i d s do not appear to explain the i r phys iological e f f ec t s , i t i s natural to consider the p o s s i b i l i t y that anaesthetics may disrupt membrane function by d i rec t interact ion with the proteins of the membrane. Anaesthetic molecules may combine with a hydrophobic region of a protein molecule, induce a conformational change and thereby make i t less act ive (Eyring et a l . 1973). A more complex hypothesis suggests that d i f f e ren t sets of hydrophobic s i tes ex i s t on target protein molecules, with each of the hydrophobic s i te s accomodating anaesthetic molecules of d i f fe rent sizes (Richards e t a l . 1978). General anaesthetics have been shown to induce changes in conformation and properties for a wide var iety of pu r i f i ed proteins and protein structures such as actomyosin (Strosberg et a l . 1972), microtubular protein of an axopod (A l l i s on et a l . 1970), glutamic dehydro-genase (Hulands e t a l . 1975), myoglobin and haemoglobin (Schoenborn and Featherstone 1967), luc i ferase (Franks and Lieb 1984; 1985), albumin and lactog lobu l in (Balasubramanian and Wetlaufer 1966), calcium-dependent ATPase (Pr ice et a l . 1974), and receptors for acety lchol ine (Young et a l . 1978; Aronstam et a l . 1986), dopamine, ouabain, or opioids (Labella et a l . 1979), as well as a-adrenergic ligands (Fairhurst and L iston 1979) and aequorine (Baker and Schapira 1980). The k inet i c s of interact ion between the anaesthetic molecules and functional proteins often indicate competitive antagonism according to Labe l l a ' s (1981) observations on anaesthetic-induced H. EL-BEHEIRY 21 pe r tu rba t i on s of s p e c i f i c b ind ing to b r a i n membranes of l i g and s f o r the o p i o i d , d i g i t a l i s and dopamine recepto r s ( L a b e l l a e t a l . 1979). Th i s i s supported by the observat ions of a halothane induced-depress ion of the p u r i f i e d c a rd i a c m y o f i b r i l l a r ATPase and the compet i t i v e antagonism by Ca of the maximal i s o m e t r i c f o r c e of i s o l a t e d c a r d i a c muscle ( P r i c e 1974; P r i c e e t a l . 1975). However, there are other r epo r t s i n d i c a t i n g non-compet i t i ve and mixed i n h i b i t i o n of enzyme a c t i v i t y by anae s t he t i c s . For example, d i e t h y l e t he r , methoxyf lu rane, t r i c h l o r o e t h y l e n e , ch l o r o f o rm and halothane show "mixed" k i n e t i c s i n i n h i b i t i n g b r a i n and e r y t h r o c y t e a c e t y l -c h o l i n e s t e r a s e a c t i v i t y (B raswel l and K i t z 1977). This d i scordance w i th the r e s u l t s of o ther s t r u c t u r a l , f u n c t i o n a l and b ind ing s t ud i e s suggest t ha t t he re are c l e a r l y d i s t i n g u i s h a b l e types of b ind ing s i t e s on p r o t e i n s . Hydrophobic pockets can r e s u l t from i n e f f i c i e n t f o l d i n g of the p r o t e i n s and can b ind anaes the t i c s w i th high s e l e c t i v i t y or cause l i t t l e or no conforma-t i o n a l change, a l though a t h igher concen t ra t i on s , anae s the t i c s can cause marked s t r u c t u r a l changes e . g . , in hemoglobin (Harkey et a l . 1979). Other hydrophobic pockets occur where p r o te i n s may f o l d around a s ub s t r a t e or c o f a c t o r ( e . g . , adenylate k i na se ; Sachsenheimer e t a l . 1977 and l u c i f e r a s e ; Franks and L ieb 1984) and there appears to be con s i de rab l y more f l e x i b i l i t y which a l lows a wider range of chemical s t r u c tu re s to b i nd . There are a l s o hydrophobic regions (or c l e f t s of c e r t a i n s i z e s ) , perhaps i n te rmed i a te between the above two, where one or more anae s the t i c s may b ind ( e . g . 6 - l a c t o g l o b u l i n ; Wishina and P inde r 1966). The hydrophobic su r f ace s or grooves where anae s the t i c s b ind i n a nonhyperbol ic p a t t e r n are l e s s d i s c r e t e and have poor l y de f ined s t o i ch i omet r y ( e . g . , bovine serum a lbumin; Wishina and P inder 1964). Whether such b ind ing r e f l e c t s a s i n g l e l a r ge patch o r many s i t e s of low a f f i n i t y has y e t to be e s t a b l i s h e d . H. EL-BEHEIRY 22 The consequences of anaes the t i c b ind ing to p r o t e i n f u n c t i o n are l e s s we l l understood. Nonethe less , three bas ic mechanisms have been proposed ( c f . M i l l e r 1985): (a) compet i t i on f o r c o f a c t o r s needed f o r a c t i v a t i o n , (b) minor s t e r i c a l l y induced rearrangements i n the t a r g e t p r o t e i n r e s u l t i n g from b ind ing to hydrophobic s i t e s near the a c t i v e cen te r and, (c) a l l o s t e r i c e f f e c t s induced by anaes thet i c p r o t e i n i n t e r a c t i o n t h a t r equ i r e anae s the t i c s to b ind to d i f f e r e n t conformat ions of a p r o t e i n w i th d i f f e r e n t a f f i n i t i e s , although t h i s has not been e s t a b l i s h e d . Two main c r i t i c i s m s have been launched aga in s t the " p r o t e i n p e r t u r b a t i o n hypothes i s " (Ueda and Kamaya 1984). The l a r ge number of p r o t e i n s t h a t show s p e c i f i c anaes the t i c b ind ing suggest t ha t n o n - s p e c i f i c i t y i s favoured. Second, the re i s v i r t u a l l y no evidence f o r the mechanism by which p r o t e i n hypothes i s can account f o r the pressure r eve r sa l phenomenon. I t i s t rue t h a t most p r o t e i n s s t ud i ed thus f a r bear no r e l a t i o n to those i n vo l ved i n synapt i c t r a n s m i s s i o n ; however, they can be cons idered as models f o r i l l u -s t r a t i n g the p o s s i b i l i t y of s p e c i f i c b ind ing of anae s t he t i c s to p r o t e i n macromolecules. Th i s s i t u a t i o n i s s i m i l a r to the use of membrane models and o l i v e o i l / w a t e r p a r t i t i o n c o e f f i c i e n t s to demonstrate the f e a s i b i l i t y of the l i p i d hypotheses. Recen t l y , however, a s p e c i f i c b ind ing of halothane to b r a i n musca r in i c r ecep to r s has been reported (Aronstam 1986). The impress i ve agreement between the p r e d i c t i o n s of the c r i t i c a l volume hypothes i s (a n o n - s p e c i f i c theory ) and the pressure r eve r sa l data cou ld be more obta ined p r e c i s e l y by assuming a s i n g l e molecu la r model i n which one anaes the t i c molecule b inds t o , and i n a c t i v a t e s a s i n g l e s i t e (Franks and L ieb 1982). In a d d i t i o n , pressure r e v e r s a l c ou l d be exp la ined by pressure caus ing a general i nc rease i n e x c i t a b i l i t y , which counteract s a g ene r a l i z ed decrease i n H. EL-BEHEIRY 23 e x c i t a b i l i t y caused by anaes thet i c b ind ing to s p e c i f i c p r o t e i n s i t e s (Kendig e t a l . 1975, 1978) or s imply t o anae s the t i c s being "squeezed away" from t h e i r t a r g e t s i t e s . 1.3.3.3 Anaes the t i c mo lecu la r mechanisms and c e l l u l a r e x c i t a b i l i t y . In o rde r to accept a molecu la r hypothes is f o r anae s the t i c a c t i o n s , the ques t ion about how l i p i d or p r o t e i n pe r t u r ba t i o n s cou l d produce depress ion i n c e l l u l a r e x c i t a b i l i t y should be addressed. Because t h i s i s d i f f i c u l t to answer, few repo r t s have attempted to exp lo re the l i n k between molecu la r mechanisms and neuronal depres s ion . Haydon and a s s o c i a t e s (1977) repor ted a decrease i n capac i tance and t h i c k e n i n g of b lack l i p i d membranes induced by a lkanes and anae s the t i c s i n a dose-dependent manner. L a t e r , the same group (Haydon and Urban 1983 a, b, c) us ing two -e l ec t rode vo l tage-c lamp techniques i n squ id axon found t h a t an i nc rea se i n the steady s t a t e i n a c t i v a t i o n of Na-channels was a s soc i a ted w i th a decrease i n input capac i tance ( i . e . , an i nc rea se in membrane t h i c k n e s s ) when an anae s the t i c was app l i ed i n the bath ing s o l u t i o n . Urban (1985), us ing the same techn iques , pub l i shed a Meyer-Overton type of c o r r e l a t i o n between anae s the t i c concen t ra t i on s needed to b lock evoked inward sodium cu r r en t s by 50%, and t h e i r membrane/buffer p a r t i t i o n c o e f f i c i e n t s . I t may be suggested t h a t t h i s i s the miss ing l i n k between the l i p i d hypotheses and the i n h i b i t i o n of e x c i t a b i l i t y . For example, anae s the t i c molecules f l u i d i z e the l i p i d membrane b i l a y e r s thereby induc ing membrane t h i c ken i n g and d e s t a b i l i z e the open i o n i c channels a c t i v a t e d du r ing e x c i t a t i o n . Franks and L i eb (1978) using X- ray d i f f r a c t i o n s t ud i e s of m u l t i l a m e l l a r l i p i d b i l a y e r s showed no change i n t h i c k n e s s f o l l o w i n g a p p l i c a t i o n s o f seve ra l a nae s t he t i c s . Fernandez e t a l . (1982) found t ha t i n the squ id axon, c h l o r o f o rm reduces the magnitude of the Na-gat ing c u r r en t s but has no e f f e c t H. EL-BEHEIRY 24 on t h e i r k i n e t i c s . In c o n t r a s t ch l o ro fo rm inc reases the r a te of charge t r a n s l o c a t i o n of the l i p o p h i l i c i on d i p i c r y l a m i n e . I t i s t he r e f o r e u n l i k e l y t h a t general anae s the t i c s ac t to modify the k i n e t i c s of opening and c l o s i n g of i o n i c channels v i a m o d i f i c a t i o n of the s t r u c t u r a l parameters of the l i p i d m a t r i x . More recent evidence showed t h a t membrane capac i tance of hippocampal neurons had been increased (not decreased) by ethanol a p p l i c a t i o n s (Car len e t a l . 1986). P u i l and Gimbarzevsky (1987) a l s o repor ted an inc rease of the i npu t membrane capac i tance i n t r i g e m i n a l root gang l i on neurons induced by halothane and i s o f l u r a n e i . e . , i n neurons t h a t are be l i e ved to be devoid of s ynap t i c i npu t s (Lieberman 1976). Hence, i f the theory of membrane t h i c k e n -ing can su r v i ve the c r i t i c i s m of e x t r a p o l a t i n g obse rva t ions from squ id axon to mammalian c e n t r a l neurons, some d i f f i c u l t y w i l l be encountered i n r e c o n c i l i n g the abovementioned observat ions t h a t are c o n t r a d i c t o r y to those d i s cu s sed above (Haydon e t a l 1977; Haydon and Urban, 1983a, b, c ) . Another attempt to b r idge the gap between the mo lecu la r hypotheses and the depres s ion of e x c i t a b i l i t y was the d i scovery of a novel " anae s the t i c K - cu r r en t " s p e c i f i c a l l y a c t i v a t e d by halothane i n mol luscan neurons and 2+ i n s e n s i t i v e to 4 amino-pyr id ine (4-AP), tetraethylammonium (TEA), Co and Na + replacement (Franks and L i eb 1988). They suggested t ha t anae s the t i c s b ind d i r e c t l y to the r e l e van t channel p r o t e i n and s t a b i l i z e i t i n the a c t i v a t e d s t a t e or a l t e r n a t i v e l y a c t a t some p o i n t on the second messenger system t h a t regu la te s the channe l . However, t h i s may not prov ide the app rop r i a t e l i n k between the p r o t e i n p e r t u r b a t i o n hypothes i s and the a t t enua t i on of membrane e x c i t a b i l i t y . A l a r g e v a r i e t y of anae s the t i c s i n c l u d i n g v o l a t i l e and in t ravenous agents and t h e i r a b i l i t i e s t o a c t i v a t e such c u r r e n t should be i n v e s t i g a t e d i n cases where t h e i r E C r n ' s are H. EL-BEHEIRY 25 s t r ong l y c o r r e l a t e d w i th t h e i r minimum a l v e o l a r concen t r a t i on (MAC) v a l ue s . In a d d i t i o n , t h i s novel anae s the t i c a c t i v a t e d K-conductance r e q u i r e s demonstrat ion i n neurons of the CNS i n ve r teb ra te an imal s . In conc l u s i on the data i n the l i t e r a t u r e does not f i l l the gap between mo lecu la r mechanisms of anaes thes ia and the anae s the t i c - i nduced depress ion of e x c i t a b i l i t y . 1.3.3.4 Anae s the t i c a c t i o n s on cytop lasmic membrane s i t e s . In the absence of a p l a u s i b l e e xp l ana t i on of how anaes the t i c s depress neurons by i n t e r a c t i n g w i th plasma membrane l i p i d s or p r o t e i n s , K r n j e v i c (1972, 1974b, 1975; 1986) hypothes ized t h a t anae s the t i c s cou ld ac t i n d i r e c t l y a t c y t o -2+ plasmic membrane s i t e s . For example, anaes the t i c s might depress the Ca 2+ accumulat ing a c t i v i t y of mi tochondr ia and other cy top lasmic Ca - b i n d i n g 2+ membranous p r o t e i n s , thus i n c r e a s i n g in t raneurona l l e v e l s of f r ee Ca , which i n tu rn cou ld i n f l u e n c e the conductance p r o p e r t i e s of e x c i t a b l e membranes as we l l as a l t e r the p re synap t i c r e l ea se of t r a n s m i t t e r s . Support f o r t h i s hypothes i s i s suggested by both b iochemical and e l e c t r o p h y s i o l o g i c ev idence: (a) i n h a l a t i o n a l agents i n h i b i t m i tochondr i a l a c t i v i t y (Brunner et a l . 1971; Biebuyck 1973; Rosenberg and Haugaard 1973; Hawkins and 2+ Biebuyck 1980). Large r educ t i on s i n m i tochondr i a l Ca uptake by in t ravenous anae s the t i c s a l s o have been observed i n i n v i t r o p repara t i ons (Lee e t a l . 1979; Sweetman e t a l . 1981) and i n i n v i v o p repa ra t i on of r a t b r a i n (Sweetman and Esmail 1975), 2+ (b) b a r b i t u r a t e s i nc rea se f r e e i n t r a c e l l u l a r Ca a c t i v i t y i n CNS neurons of ca t s i n v i vo (Mor r i s and K r n j e v i c 1985; M o r r i s e t a l . 1986). In b r a i n synaptosomes severa l i n h a l a t i o n a l a n a e s t h e t i c s , i n 2+ h igher doses, i n c rea se the i n t r a c e l l u l a r f r e e Ca c o n c e n t r a t i o n ( [ C a 2 * ^ . ; D a n i e l l and H a r r i s 1988), H. EL-BEHE IRY 26 (c) 2 , 4 - d i n i t r opheno l (DNP), a metabol ic i n h i b i t o r , produces e l e c t r o -p h y s i o l o g i c a l e f f e c t s on c e n t r a l neurons s i m i l a r to those of anaes-t h e t i c s , p a r t i c u l a r l y the a l t e r e d respons iveness to e x c i t a t o r y amino ac id s and pept ides (Godfra ind et a l . 1970; 1971; Ca tch love e t a l . 1972; K r n j e v i c e t a l . 1978a; Sa s t r y 1978), d) hypoxia i n i n v i vo or i n v i t r o p repara t i ons induces e x c i t a b i l i t y changes s i m i l a r t o anae s the t i c a c t i on (G lo tzner 1967; LeBlond and K r n j e v i c 1989; K r n j e v i 6 and LeBlond 1989) and (e) more r e c e n t l y , K r n j e v i c and P u i l (1988) showed t h a t halothane i n 2+ c l i n i c a l l y r e l e van t concen t ra t i on s depressed inward Ca c u r r en t s i n hippocampal CA 1 neurons i n a dose-dependent manner w i thout any e f f e c t on the leak conductances. A l though i t seems t h a t t h i s e legant hypothes i s might e x p l a i n the c e l l u l a r bas i s of anae s the t i c a c t i o n s , there are severa l c r i t i c a l p o i n t s t h a t remain unanswered. For example, ha lothane- induced depres s ion of m i tochondr i a l r e s p i r a t i o n i s not reversed by high pressure (Cohen 1973). The quest ion of how anae s the t i c s ac t on m i tochondr i a l membranes (or o ther Ca-b ind ing c y t o s o l i c p r o t e i n s ) has not been re so l ved by exper imenta l ev idence, i . e . , anae s the t i c molecules a l t e r these membrane s t r u c t u r e s by unknown b i o p h y s i c a l mechanisms ( K r n j e v i c 1974b). The e l e v a t e d [Ca 2 * ] . . was suggested to s u b s t a n t i a l l y i nc rease the input conductances of the neurons induc ing h y p e r p o l a r i z a t i o n secondary to an enhanced K + - e f f l u x . Anaes the t i c s do not c o n s i s t e n t l y evoke h y p e r p o l a r i z a t i o n even i n the same neuronal type ( e . g . , hippocampal CA1 neurons; Berg-Johnsen and Langmoen 1987; Fu j iwa ra et a l . 1988; Miu and P u i l 1989). Acceptance of t h i s hypothes i s w i l l depend on f u r t h e r e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n s of a s i n g l e c e l l type us ing a v a r i e t y of i n t ravenous and i n h a l a t i o n a l a n a e s t h e t i c s , as we l l as a sys temat ic b iochemica l approach to H. EL-BEHEIRY 27 e l u c i d a t e the e f f e c t s of the spectrum of anae s t he t i c s on the r e s p i r a t o r y 2+ cha i n enzyme system combined w i th [Ca measurements i n both i n v i vo and i n v i t r o mammalian p r epa r a t i on s . Such i n v e s t i g a t i o n s should be c a r r i e d out i n nervous t i s s u e r e l e van t to the n e u r o l o g i c a l mechanisms of con sc i ou s -ness d i s cu s sed above. 1.4 Hypotheses and o b j e c t i v e s . The primary o b j e c t i v e of the present i n v e s t i g a t i o n s was to study the neuronal mechanisms by which anae s the t i c s depress n e o c o r t i c a l a r o u s a l . However, t h i s o b j e c t i v e cou ld not have been a t t a i n e d unless three bas ic quest ions were addressed: 1. What are the e f f e c t s of anaes thet i c agents on the neuronal e x c i t a -b i l i t y of n e o c o r t i c a l neurons? 2. Is the anae s the t i c depress ion of neocortex due to po ten t i a t ed i n h i b i t i o n or depressed e x c i t a t i o n ? 3. What are the p l a u s i b l e i o n i c mechanisms of the anaes the t i c ac t i on s ? During the course of the i n v e s t i g a t i o n s , a major hypothes is developed suggest ing t h a t c h o l i n e r g i c e x c i t a t i o n i n the neocortex i s more vu lne rab le t o depres s ion by anae s t he t i c s than g lu tamate rg i c s t imu l a t o r y e f f e c t s . A c c o r d i n g l y , an ex ten s i ve study was designed t o determine the e f f e c t s of anae s the t i c s on the chemica l s e n s i t i v i t i e s of n e o c o r t i c a l neurons to a p p l i e d t r a n s m i t t e r substances. 2 METHODS 2.1 Animals Duncan H a r t l e y guinea p igs (males or females) were obta ined from the Animal Care Centre of the U n i v e r s i t y of B r i t i s h Columbia. The guinea p igs were weaned a t t h i s Cen t re , from l a c t a t i n g females a f t e r 14 days, and fed on H. EL-BEHEIRY 28 a v i t am in C-supplemented chow, wi th f r e e access to water . Once a week, 5-6 guinea p ig s (150-250 g, or approx imately 20 days o ld) were rece i ved from the Animal Un i t and p laced in a wire cage i n the animal care room of the Department of Pharmacology & Therapeut i c s f o r l e s s than one week before an exper iment. The temperatures and humidity i n these f a c i l i t i e s were c o n t r o l -l ed a t 22-23°C and 50-55%, r e s p e c t i v e l y . 2.2 S l i c e p repa ra t i on s Anaes thes i a was induced w i t h d i e t h y l e t h e r and mainta ined by endotracheal adm in s t r a t i on of 1-2% halothane. Attempts to lower i n t r a c r a n i a l t en s i on were made w i t h a r t i f i c i a l v e n t i l a t i o n by c o n t r o l l i n g the e n d - t i d a l CO,, c o n c e n t r a t i o n at 30-35 mm Hg. A f t e r f rontotempora l cran iotomy, the dura was r e f l e c t e d to expose the neocortex. In view of the v u l n e r a b i l i t y of n e o c o r t i c a l neurons to hypoxia and o the r derangements of metabol i sm, the b lood supply of the b r a i n was u n i n t e r r u p t e d up to the moment of the c o r t i c a l e x c i s i o n . The sensor imotor and the a n t e r i o r c i n g u l a t e c o r t i c e s were i d e n t i f i e d ( Z e i g l e r 1964; Verne 1974), e x c i s ed w i th some cont iguous s u b c o r t i c a l t i s s u e , and immersed i n c o l d (2°C) a r t i f i c i a l c e reb ro sp i na l f l u i d (ACSF) t h a t was oxygenated w i t h a 95/5% gaseous mixture of 0 2 / C 0 2 (pH 7.4) w i t h i n 10 s of e x c i s i o n . The c o n s t i t u e n t s of the ACSF were ( i n mM): NaC l , 124; KC1, 3.75; K H 2 P 0 4 , 1.25; MgS0 4 .7H 2 0, 2; C a C l 2 . 2 H 2 0 2; dex t r o s e , 10; NaHC0 3, 26. The t i s s u e was trimmed i n t o ~5 mm square b l o c k s , g lued w i th a -cyanoacrylate to a T e f l o n d i s c and cu t c o r o n a l l y w i t h a V i b r o s l i c e r i n t o 500-600 nm t h i c k s l i c e s . Dur ing the s l i c i n g procedure which l a s t e d a t o t a l of 15-20 min, the t i s s u e was immersed i n con t i nuou s l y oxygenated ACSF a t ~4°C. The s l i c e s were t r a n s f e r r e d to a chamber c on t a i n i n g oxygenated ACSF at room temperature (~22°C) and a l lowed to warm up g r a d u a l l y f o r a t l e a s t 30 min. Each b lock of H. EL-BEHEIRY 29 t i s s u e y i e l d e d ~5 n e o c o r t i c a l s l i c e s together w i th some cont iguous subcor-t i c a l s t r u c t u r e s . A s l i c e was t r a n s f e r r e d to the record ing chamber (1 ml volume, F i g . 1) con t i nuou s l y superfused wi th ACSF i n a hum id i f i ed oxygenated atmosphere (Pandanaboina and Sast ry 1984). Here, the temperature was con -t r o l l e d at 32-34°C wi th a thermoregu la to r . A nylon mesh prevented f l o a t i n g movement of the s l i c e s dur ing pe r f u s i o n ( r a t e 2-4 ml/min). 2.3 S o l u t i o n s and drugs A l t h e s i n (G laxo ) , which i s a 3:1 mixture of two s t e r o i d s , a lphaxa lone and alphadolone in 20% v/v po l yoxye thy l a ted ca s t o r o i l (cremophor EL ) , was d i l u t e d on the day of the experiment w i t h the ACSF t o o b t a i n the de s i r ed c oncen t r a t i o n s . The concen t r a t i on s of the v e h i c l e in the s o l u t i o n s used i n -5 -5 these i n v e s t i g a t i o n s ranged between 2 x 10 and 16 x 10 % v/v. P rev iou s i n v e s t i g a t i o n s on the anae s the t i c potenc ies of a lphaxa lone i n the absence and presence of cremophor E l have a s ce r t a i ned t h a t the observed a c t i v i t i e s of A l t h e s i n on neuronal membranes are a t t r i b u t a b l e main ly t o t h i s s t e r o i d , and not to the v e h i c l e (Pennefather e t a l . 1980; Cu l l en and M a r t i n 1982, M inch in 1981). Before a p p l i c a t i o n by p e r f u s i o n , i s o f l u r a n e (Anaquest) was vapor i zed wi th the 9 5 / 5 % , O g / ^ gaseous mixture us ing f i v e Mark 3 F l u o t e c vapo r i ze r s (Cyprane, K e i g h l e y , UK) t h a t were r e c e n t l y c a l i b r a t e d , and bubbled i n t o the ACSF r e s e r v o i r s ( i n v e r t e d and covered 60 ml s y r i nges ) f o r a minimum of 20 min us ing a d i l u e n t gas f l ow rate of 1 L/min ( F i g . 1 ) . I s o f l u r ane vapor a l s o was i n t roduced a t the des ignated c o n c e n t r a t i o n i n t o the chamber atmosphere immediately above the submersed s l i c e . The concen-19 t r a t i o n of i s o f l u r a n e i n the bath was determined us ing f l u o r i n e - n u c l e a r magnetic resonance techn iques ( c f . Miu and P u i l 1989). The molar va lues thereby obta ined were conver ted to t h e i r corresponding MAC values f o r rodents Fig. 1 Anaesthetic delivery system and recording chamber used in these investigations. co o H. EL-BEHEIRY 31 (Steward e t a l . 1973; Cu l l en 1986; F i r e s t one e t a l . 1986). A l i q u o t s of ( - ) b i c u c u l l i n e meth iod ide (Sigma) and (± )bac lofen (C iba-Ge igy ) were added to the ACSF from stock s o l u t i o n s (0.1 mM) t h a t were kept f r o zen u n t i l r equ i r ed . 2+ 2+ Ca - d e f i c i e n t s o l u t i o n s were made wi thout Ca and e i t h e r by adding 2+ 2 mM C o C l 2 or by i n c r e a s i n g the [Mg ] to 4 mM, accord ing to the exper-2+ imental procedure. In s tud ie s on the e f f e c t s of e x t r a c e l l u l a r [Mg ] 2 + ([Mg ] Q ) , the t o t a l d i v a l e n t c a t i o n c oncen t r a t i o n i n the ACSF was kept 2+ 2 + cons tant by i n c r e a s i n g or decreas ing the [Ca ] wherever the [Mg ] was changed. The o s m o l a l i t y of the s o l u t i o n s was mainta ined approx imately cons tant at 320 mosmol, by the a d d i t i o n of sucrose where necessary. 2.4 I n t r a c e l l u l a r record ing arrangement I n t r a c e l l u l a r reco rd ing s began approx imately 30 min a f t e r i ncuba t i ng a s l i c e i n the reco rd ing chamber. A m i c roe lec t rode mounted on a Narashige m ic roman ipu la to r , was po s i t i o ned under v i s u a l guidance us ing a d i s s e c t i n g microscope (40x) , approx imate ly at the middle and lower t h i r d p o r t i o n of the s l i c e ( c f . F i g . 2a) . I n t r a c e l l u l a r v o l t a ge record ings from neurons were obta ined using m i c r oe l e c t r ode s f i l l e d w i th 3 M K C l , 3 M C s 2 S 0 4 , 3 M K -acetate or 0.6 M ^ S O ^ . The t i p r e s i s t a n c e s measured in the t i s s u e were 50 to 90 Mft. The e l e c t r o d e s were p u l l e d from m i c r o f i l a m e n t - i n c o r p o r a t e d g l a s s blanks (1.0 and 1.2 mm ex te rna l d iameter ; WPI Instruments) w i th a v e r t i c a l p u l l e r (Narashige AP 81) us ing h igh heat and magnet-power s e t t i n g s . Each e l e c t r ode was checked us ing high m a g n i f i c a t i o n l i g h t microscopy f o r i t s o v e r a l l form and p r o j e c t e d t i p s i z e . The e l e c t r ode s then were v e r t i c a l l y p o s i t i o n e d on a r e t a i n i n g r i n g w i th the basal end immersed overn i gh t i n the concent rated e l e c t r o l y t e s o l u t i o n . H. EL-BEHEIRY 32 Glued Iontophoretic E l e c t r o d e Electrode 20° / Both Slice Fig. 2 Diagrams showing the neocortical s l ice and sites of recording and stimulation (a) and types of iontophoretic electrodes used (b and c ) . H. EL-BEHEIRY 33 The input r e s i s t a n c e s (R^) of the neurons were measured u s ing i n t r a c e l l u l a r i n j e c t i o n s of s p e c i f i e d step c u r r en t s v i a the b r i d ge -ba l ance c i r c u i t of a WPI a m p l i f i e r (model M701). A s e r i e s of sp ikes and AHPs were e l i c i t e d a t 0.1 Hz by i n t r a c e l l u l a r i n j e c t i o n s of c u r r e n t pu l ses be f o r e , du r i n g , and a f t e r an anae s t he t i c a p p l i c a t i o n ; each of these pu l ses evoked 5 t o 6 s p i ke s . A t o t a l of 6 l o n g - l a s t i n g AHPs were sampled c o n s e c u t i v e l y f o r each exper imental c o n d i t i o n . These were q u a n t i f i e d by averag ing t h e i r peak ampl i tudes and du r a t i on s . Because AHP parameters such as ampl i tude and du ra t i on can be a f f e c t e d by changes i n r e s t i n g membrane p o t e n t i a l (Vm)> cont inuous DC - i n j e c t i o n s were used to compensate f o r any anae s the t i c induced changes i n V m . A tungsten b i p o l a r s t i m u l a t i n g e l e c t r o d e was p laced i n the subp i a l reg ion of the n e o c o r t i c a l s l i c e f o r s tudy ing the e f f e c t s of anaes-t h e t i c s on synapt ic p o t e n t i a l s ( F i g . 2a). S t i m u l a t i o n frequency was set to 0.2 Hz and the s t imu lu s s t r eng th ranged from 5-10 V. The a m p l i f i e d p o t e n t i a l s were d i g i t i z e d with a PCM-1 i n s t r umenta t i on u n i t (VCR adaptor ; Medica l Systems Inc.) and s to red w i t h a super VHS v ideo tape reco rder (Canon model VR-HF 600) f o r o f f - l i n e playback and a n a l y s i s by computer. In some cases the i n t r a c e l l u l a r s i g na l s ( i n analogue form) were ana lyzed wi th a waveform reco rde r (Biomation 805) which d i g i t i z e s the i nput vo l t age sweep i n t o 2048 po i n t s and thereby permits manual c o n t r o l of the d i g i t a l - t o - a n a l o g u e conver s ion and storage f o r d i s p l a y on the o s c i l l o s c o p e , o r rep roduc t i on on paper i n an X-Y pen p l o t t e r (Hewlett -Packard 7015B; F i g . 3 ) . 2.5 Computer a n a l y s i s I n t r a c e l l u l a r vo l t age s i g n a l s , p layed back from the VHS v ideo r e co rde r , were fed i n t o an i n t e r f a c e panel of a data a c q u i s i t i o n system (ERAT; l a b o r a t o r y - b u i l t ) which conver t s the i nput analogue s i g n a l s to d i g i t a l form S t i m u l a t o r Sunn ing f ^ A m p l i f i e r v * WPI A n p l i f i e r WPI P r o b e DC Bias • s c i l l o s c o p e c VCR „ PCM-1 / Biomatlon / XY P l o t t e r I n t e r f a c e ERAT MINC 23 PDP 11/44 Tern ina l Fig. 3 Organization of the experimental set-up for data display, acquisition and retrieval. H. EL-BEHEIRY 35 (A/D conve r s i o n ) . The data were s tored t empo ra r i l y i n the memory of a MINC-23 computer (DEC Inc.) during the data a c q u i s i t i o n . A f t e r complet ion of t h i s phase, the f i l e s were t r a n s f e r r e d and s to red f o r the averaging and o the r procedures i n another computer (PDP 11/44; DEC Inc.) which has a l a r g e r memory c apac i t y ( F i g . 3 ) . F i f t e e n to twenty i n d i v i d u a l t r ace s of the synapt i c t r a n s i e n t s i n the raw data were s e l e c t ed from a MINC f i l e f o r an averaging procedure. A new f i l e was subsequent ly c rea ted f o r the averaged t r a c e s , and the ampl i tude, d u r a t i o n , r a te of decay of the synapt i c t r a n s i e n t s were measured from these averaged t r a c e s . 2.6 I on topho re t i c techniques 2.6.1 E l e c t r o d e s . In the i n i t i a l exper iments, 5 b a r r e l l e d i on t opho re t i c e l e c t r o d e s w i t h a common t i p diameter of 9-12 nm were g lued w i th an u l t r a v i o l e t (u. v.) s e n s i t i v e f l u i d adhesive ( L o c t i t e g la s s bond or L o c t i t e 358; L o c t i t e U.K. L t d . ) t o the record ing e l e c t r o d e s ( r e s i s t a n c e s , 70-90 Mn) a t an angle of 20-30° and an i n t e r t i p d i s t ance of 30-60 \im ( K r n j e v i c and Schwartz 1967; Z ieg l gansberge r and P u i l 1973). The i o n t o p h o r e t i c p i p e t t e was bent w i t h a m ic ro fo rge a t a po i n t ~4 mm from the t i p a t an angle of 2 0 - 3 0 ° . The ' h o c k e y - s t i c k ' shaped p i p e t t e then was p o s i t i o n e d h o r i z o n t a l l y w i t h two microscopes (one i n the X-Y p lane and the o ther i n the X-2 plane) such t h a t the ' b l a d e ' of the hockey s t i c k was p a r a l l e l to the record ing m i c r o e l e c t r o d e . The bonding ma te r i a l was a p p l i e d and subjected to u.v. l i g h t f o r ~3 min w i th a Nuva L i t e gun (Caulk I n c . ) . Secondary s t rengthen ing of the e l e c t r o d e assembly was achieved by g l u i ng a g la s s tube across the two e l e c t r ode s or by app l y i ng a r a p i d l y s e t t i n g denta l wax ( F i g . 2b) at about 0.5 - 1 cm from the t i p s . Th i s assembly ensured t h a t the t r a n s m i t t e r substances would be a p p l i e d i n the v i c i n i t y of the soma of an impaled neuron. H. EL-BEHEIRY 36 In l a t e r exper iments , separate reco rd ing and i o n t o p h o r e t i c e l e c t r ode s were used i n combinat ion w i t h TTX a p p l i c a t i o n to e l i m i n a t e the e f f e c t s on nearby neurons a c t i v a t e d by an e j e c ted t r a n s m i t t e r substance. In o rder to p o s i t i o n the i o n t o p h o r e t i c e l e c t r o d e t i p as near as p o s s i b l e to an impaled neuron, i t was i n i t i a l l y assumed t h a t the impaled neuron was l o c a t e d at a d i s t ance of ~200 um from the su r face of the s l i c e . S i nce the i on t opho re t i c e l e c t r o d e was p o s i t i o n e d at an angle of 20° w i th the h o r i z o n t a l p l a n e , t r i g o n o m e t r i c f u n c t i o n s were used to determine the amount of t r a v e l f o r the i o n t o p h o r e t i c e l e c t r o d e i n t o the s l i c e ( c f . F i g . 2C). This twin e l e c t r o d e placement permi t ted a p p l i c a t i o n s of the t r a n s m i t t e r s to the dend r i t e s of the neurons. Once a neuron was impaled, Glu was e j e c t e d wi th a con t inuous l y a p p l i e d c u r r e n t (~80 nA); the " a p p l i c a t i o n e l e c t r o d e " then was advanced very s l owly w i th an independent micromanipu lator u n t i l a maximal d e p o l a r i z a t i o n was observed. 2.6.2 S o l u t i o n s and equipment. Four b a r r e l s u s u a l l y were f i l l e d using a 32 gauge needle w i th the f o l l o w i n g s o l u t i o n s of drugs (most ly from Sigma): a c e t y l c h o l i n e Cl (0.5 M, pH 4 ) , Na S-g lutamate (1 M, pH 8 . 5 ) , NMDA (200 mM, pH 9 ) , GABA (0.5 M, pH 3.5) and (±)bac lofen (C iba -Ge igy ; 50 mM, pH 3 ) . The f i f t h b a r r e l was f i l l e d w i t h a c o n t r o l s o l u t i o n (NaCl , 10 mM) which was used to compensate f o r the e j e c t i o n c u r r e n t s ; t h i s ba l anc ing channel a l s o served as a c o n t r o l f o r e f f e c t s of c u r r e n t s on neurons. The IDS 100 m i c ro i on topho re s i s u n i t used i n these experiments possessed two main f e a t u r e s . I t a l lowed the automatic use of a ' p u s h - p u l l ' technique i n which a c u r r e n t oppos i te i n p o l a r i t y and equal i n magnitude and du ra t i on c ou l d be passed through the NaC l - con ta i n i ng b a r r e l s imu l taneous l y w i t h the " d r u g - c u r r e n t " and secondly , i t had a b u i l t - i n sequencer by which the t r a n s m i t t e r substances c ou l d be a p p l i e d s e q u e n t i a l l y i n a p a t t e r n a t de s i r ed ( p re se t ) c u r r en t s f o r c e r t a i n du ra t ions and i n t e r - a p p l i c a t i o n i n t e r v a l s . H. EL-BEHEIRY 37 2.6.3 Exper imental procedures. The e f f e c t s of a nae s t he t i c s were assessed using the f o l l o w i n g procedures: (1) a f t e r s t ab l e reco rd ing c o n d i t i o n s were ach ieved, the i o n t o p h o r e t i c c u r r e n t was adjus ted i n the range of 30-350 nA ( t y p i c a l l y 80 nA) such t h a t a submaximal ( ED50) or a jus t -max imal response cou ld be e l i c i t e d w i t h a t r a n s m i t t e r substance; (2) responses of r e l a t i v e l y cons tant ampl i tude ( u s u a l l y 10-20 mV) were evoked at app rop r i a te i n t e r v a l s (10-30s) f o r a pe r i od of at l e a s t 5-8 min; (3) equ i -ampl i tude responses to a t l e a s t two t r a n s m i t t e r substances were evoked i n the same neuron; (4) pe r f u s i on of the anae s the t i c was commenced dur ing the c o n t i n u i n g , i n t e r m i t t e n t t r a n s m i t t e r a p p l i c a t i o n s , and (5) a t l e a s t 3 or 4 responses to each t r a n s m i t t e r substance obta ined j u s t before anae s the t i c a p p l i c a t i o n , a f t e r g iven i n t e r v a l s of anae s the s i a , and dur ing the recovery p e r i o d , were averaged f o r the q u a n t i t a t i v e comparisons. A s l i g h t l y d i f f e r e n t procedure was used f o r i n v e s t i g a t i o n of NMDA responses because of the r a p i d development of d e s e n s i t i z a t i o n or t achy -p h y l a x i s i n the l a r g e pyramidal ( l a y e r s IV-V) neurons ( c f . l a y e r s 11-111 i n 2+ Thomson, 1986) to a low dose of NMDA i n the presence of [Mg ] . The jus t -max imal e f f e c t was determined us ing i n f r equen t (~3 min) a p p l i c a t i o n s o f shor t d u r a t i o n . Then, 3 or 4 equ i - amp l i tude responses were obta ined a t 3 min i n t e r v a l s before commencing anae s the t i c a p p l i c a t i o n f o r ~9 min. 2+ 2.7 I n t r a c e l l u l a r Ca measurement 2.7.1 Neuronal c u l t u r e s . Hippocampi were d i s s e c ted out from 18-day-o ld W i s ta r r a t embryos. C e l l s were d i s s o c i a t e d , put onto 18mm c o v e r s l i p s and H. EL-BEHEIRY 38 incubated f o r 2 weeks i n 5% C 0 2 a t 37°C i n Du lbecco ' s mod i f i ed medium which c o n s t i u t e d 6 gm/L dex t r o se , 10 mM HEPES and 10% horse serum. 2.7.2 Storage and l oad ing of Fura-2 AM. Fura-2 AM (1 mg; Mo lecu l a r Probes Inc.) was d i s s o l v e d i n ch lo ro fo rm and 50 LIL a l i q u o t s which were p i p e t t e d i n t o small p l a s t i c ampules, p laced i n a d e s s i c a t o r and vacuum-dried f o r 3 hours. The d r i e d a l i q u o t s were s tored at -80°C. On the day of the experiment 50 ug of Fura-2 was d i s s o l v e d i n 50 uL d imethyl s u l f o x i d e (DMSO) y i e l d i n g stock s o l u t i o n s of ~1 mM. Stock Fura-2 (10 was added to 2 ml pre incubated E a r l ' s balanced s a l t s o l u t i o n (EBSS) y i e l d i n g a c o n c e n t r a t i o n o f 10 uM Fura -2 . EBSS c o n s t i t u t e d i n mM: MgS0 4 < H 2 0, 0 .8 ; K 2 S 0 4 , 2.7; NaC l , 117; NaHC0 3, 26; NaH 2 P0 4 .H 2 0 , 1; dex t r o se , , 5.6; HEPES, 10; C a C l 2 . H 2 0 , 1.8 and 0.5% phenol red i n d i c a t o r (1 ml/L EBSS). One ml of EBSS-conta in ing Fura-2 was immediately added to the we l l w i th 1 ml of standard EBSS; the hippocampal c e l l c u l t u r e then was p laced face-up i n the we l l and incubated f o r 2 hours. Before r e co rd i ng , the Fura-2 incubated c u l t u r e was r i n s ed by p l a c i n g i t i n t o a separate d i sh of standard EBSS and incubated a f u r t h e r h a l f - h o u r a f t e r which the c e l l s were ready to use. 2+ 2.7.3 I n t r a c e l l u l a r Ca measurement. C o v e r - s l i p s c on ta i n i n g dye- loaded c e l l s were i n ve r t ed and sealed i n t o a s p e c i f i c a l l y designed record ing chamber a l l o w i n g 2 pe r f u s i o n input s - - one f o r the c o n t r o l s o l u t i o n , low c h l o r i d e EBSS i n which NaCl was s u b s t i t u t e d f o r 117 mM Na i s e t h i o n a t e , and the o the r f o r admin s t r a t i on of a nae s t he t i c s . A t h i r d port was used f o r the i n j e c t i o n of ul -volumes of the t r a n s m i t t e r substances and agon i s t s . S t i m u l a t i o n of the NMDA-receptor subtype by g lutamate was favoured by adding g l y c i n e (1-2 LIM) and exc l u s i on of Mg dur ing the p repa ra t i on of the low c h l o r i d e EBSS. A c t i v a t i o n of qu i s qua l a t e (Quis ) - subtype of r ecep to r was achieved by us ing low c h l o r i d e EBSS w i thout H. EL-BEHEIRY 39 the e x c l u s i o n of Mg. Continuous suc t ion was a p p l i e d to an oppos i te opening and the temperature was c o n t r o l l e d by a regu la ted heat ing dev i ce beneath the chamber. The c e l l s were viewed under o i l immersion a t lOOx m a g n i f i c a t i o n on a Jenalumar Z e i s s e p i f l u o r e s c e n t microscope which had an ad ju s t ab l e aper ture f o r v iewing s i n g l e neurons. Recording of f l u o r e s cence from p e r i k a r y a of s i n g l e neurons was c a r r i e d out at a l t e r n a t i n g e x c i t a t i o n wavelengths of 350 and 380 nm u.v. l i g h t . F luorescence s i g na l s (emit ted from a neuron) was passed through a G125 g l a s s b a r r i e r f i l t e r to reduce any background f l uo re s cence and then to a p h o t o m u l t i p l i e r which converted the f l uo re scence i n t o DC-vo l tage. These s i gna l s were converted to d i g i t a l form by an A/D conve r te r and s to red on f l oppy d i s c s w i t h a microcomputer. The r e s u l t s were presented as two values represent ing the i n t e n s i t y of emiss ions of both Ca - f r ee -Fu ra - 2 and Ca-bound-Fura-2 a t wavelengths o f 380 and 350 nm, 2+ r e s p e c t i v e l y . The a c t ua l [Ca ] . then was c a l c u l a t e d us ing the f o l l o w i n g fo rmu la : CCa 2 *],. . K d 2 . 8 B(R-R m i n)/«W R ) ( 1 ) where i s the r a t e cons tant of a s s o c i a t i o n o f the Fura-2 w i t h the 2+ c y t o s o l i c f r e e Ca , 8 i s the r a t i o of f l u o r e s c e n t va lues a t 380 nm w i t h zero and i n f i n i t e 2+ 2+ e x t r a c e l l u l a r Ca c o n c e n t r a t i o n ([Ca ] Q ) , R i s the e xpe r imen ta l l y determined r a t i o of the values of emiss ion from the c e l l a t 350 and 380 nm, 2+ R m i n 1 S t n e r a t l ° °f values at 350 and 380 nm at zero [Ca ] Q , 2+ R m a v i s the r a t i o of values at 350 and 380 nm at i n f i n i t e [Ca ] . niaA 0 and H. EL-BEHEIRY 40 B ' R m i n a n d Rmax a r e determined from c a l i b r a t i o n of the system. 2.7.4 C a l i b r a t i o n of the system. The f o l l o w i n g c a l i b r a t i o n procedure was employed: Two hippocampal Fura-2 loaded c e l l c u l t u r e s were e q u i l i b r a t e d w i t h the C a 2 + - i o n o p h o r e A 23187 (10 nM), the f i r s t i n the presence of 10 mM EGTA 2+ ( ca l c i um c h e l a t o r ) and zero [Ca ] , and the second i n the presence of 2 + 2 + normal [Ca ] Q and w i thout EGTA. [Ca ] . measurements were obta ined from both c u l t u r e s f o r several c e l l s u n t i l a ' p l a t e a u ' of c o n s i s t e n t values was reached. The 6 , R„,-„ and R„,„ cons tant s then were c a l c u l a t e d from mi n max the averaged va lues obta ined i n these exper iments. 2.8 S t a t i s t i c a l a n a l y s i s The e l e c t r o p h y s i o l o g i c a l r e s u l t s were subjected to t e s t s w i t h ANOVA f o r s t a t i s t i c a l s i g n i f i c a n c e . M u l t i p l e comparisons between two groups were made us ing the Mann Whitney U- tes t (Zar 1984). Sample s i z e i n a l l experiments was u s u a l l y 5 un less otherwise s t a t e d . The accepted l e v e l of s i g n i f i c a n c e was p<0.05. Regress ion a n a l y s i s was used to f i t the da ta . 3 SPONTANEOUS ACTIVITY, REPETITIVE FIRING AND  PASSIVE MEMBRANE PROPERTIES 3.1 Re su l t s The 64 neurons s e l e c t ed f o r the study of anae s the t i c a p p l i c a t i o n s on parameters of neuronal e x c i t a b i l i t y had r e s t i n g p o t e n t i a l s more negat ive than -60 mV, sp i ke ampl i tudes u s u a l l y of 70-75 mV, and an a b i l i t y to f i r e 5 such sp ikes r e p e t i t i v e l y dur ing i n t r a c e l l u l a r c u r r e n t - p u l s e i n j e c t i o n . The depths of s ucce s s fu l impalements corresponded to l a y e r s IV and V of t y p i c a l c o r t e x . The input r e s i s t a n c e s (mean * SD; 6 5 . 7 > 30.7 Mn) and r e s t i n g p o t e n t i a l s (Vm; -71.6 * 9.8 mV) remained s t a b l e f o r pe r i ods of 30 t o H. EL-BEHEIRY 41 240 min. F o r t y - f i v e a p p l i c a t i o n s of i s o f l u r a n e in doses t h a t ranged from 0.5 to 2.5 MAC were made to 34 neurons. A l t h e s i n was app l i ed 48 t imes to 30 neurons, i n concen t ra t i on s from 15 nM to 1500 uM (a lphaxalone c o n t e n t ) . The v e h i c l e , cremophor EL d i d not have any e f f e c t when te s ted on the pa s s i ve and a c t i v e membrane p r o p e r t i e s as we l l as the spontaneous a c t i v i t i e s of neurons. In the i n i t i a l i n v e s t i g a t i o n s on 8 of the neurons, 1 MAC i s o f l u r a n e or 100 uM A l t h e s i n was a p p l i e d con t i nuous l y f o r 18 min. The maximal e f f e c t s were e v i den t a t 6-9 min w i t h i s o f l u r a n e and a t 4-6 min w i t h A l t h e s i n , depending on the r a te of p e r f u s i o n . There fo re , the r a te was adjusted to permit sampling of the maximal changes i n parameters induced by e i t h e r agent at 6 min. Halothane was app l i ed 18 t imes to 10 neurons. No d i f f e r e n c e in anae s the t i c e f f e c t s was observed between the d i f f e r e n t types of sensor imotor and a n t e r i o r c i n g u l a t e neurons t h a t have been c l a s s i f i e d i n t o t h ree c a t e g o r i e s (McCormick and P r i n c e 1986). 3.1.1 E f f e c t s on spontaneous a c t i v i t y and evoked r e p e t i t i v e f i r i n g . Twenty - f i ve percent ( i . e . , 16/64) of the neurons were spontaneously a c t i v e ( s y n a p t i c t r a n s i e n t s and sp ikes ) and a r r h y t h m i c a l l y f i r e d sp i ke s . A p p l i c a t i o n s of an anae s the t i c ( F i g . 4) e i t h e r had no e f f e c t or depressed spontaneous f i r i n g ( i s o f l u r a n e , 10/12 and A l t h e s i n , 5/8 neurons). An i n i t i a l p o t e n t i a t i o n of f i r i n g f o l l owed by i n h i b i t i o n o f spontaneous a c t i v i t y was not e v i d e n t , as a l s o found i n hippocampal neurons i n v i t r o (Berg-Johnsen and Langmoen 1987). F u l l recovery u s u a l l y was observed w i t h i n 6-10 min a f t e r an a p p l i c a t i o n . O c c a s i o n a l l y , recovery was preceded by a rebound inc rease i n f i r i n g . Complete recovery was not observed i n some cases even a f t e r about 30 min ( F i g . 4 A ) . However, most other anae s the t i c induced changes were f u l l y r e v e r s i b l e . H. EL-BEHEIRY 42 IILI11L. Isoflurane H M A C I H U L L Isoflurane I2 .SMACI Althes in I100/1MI jiunN J i n i ni 5mV imin B control I F L I M A C 20 mV 0 . 5 n A recovery L A F i g . 4 I s o f l u rane and A l t h e s i n induced depress ion of spontaneous sp ike a c t i v i t i e s . (A) i s o f l u r a n e a p p l i c a t i o n (1 MAC; upper t r a ce ) produced spontaneous f i r i n g and a rebound i nc rea se i n f i r i n g a f t e r I s o f l u r a n e (2.5 MAC; middle t race ) i n the same neuron h y p e r p o l a r i z a t i o n (~3 mV) and a depress ion o f spontaneous p a r t l y r e v e r s i b l e . A l t h e s i n (100 LIM; lower t r ace ) induced a s s o c i a t e d w i th complete a t t enua t i on of spontaneous neuron. (B) on a f a s t e r t ime base, i s o f l u r a n e i n h i b i t i o n of the the a p p l i c a t i o n , produced a s l i g h t a c t i v i t y t ha t was ~5 mV h y p e r p o l a r i z a t i o n a c t i v i t y i n a second depressed spontaneous sp i kes and b locked the AHPs. Voltage responses to h y p e r p o l a r i z i n g c u r r en t pu l ses may be seen a t the l e f t of the anodal break s p i ke s . Complete recovery was observed a f t e r 10 min. There was no change i n the V m of t h i s neuron. H. EL-BEHEIRY 43 The s i n g l e spike t h r e s h o l d t e s t e d i n 62 neurons by c u r r e n t i n j e c t i o n was not a l t e r e d s i g n i f i c a n t l y i n (~70%) of cases. However, the amount of c u r r e n t r equ i red to evoke 3 to 5 sp ikes was on ly e f f e c t i v e i n produc ing <2 sp i ke s dur ing a p p l i c a t i o n s of i s o f l u r a n e (23/32 neurons) i n doses of 1 to 2.5 MAC o r A l t h e s i n (17/30 neurons) i n doses of 100-1500 uM ( F i g . 5A). A l t h e s i n a d m i n i s t r a t i o n a t the 15 o r 50 nM doses d id not produce i n h i b i t i o n o f evoked r e p e t i t i v e f i r i n g ( F i g . 5B). Hence, i s o f l u r a n e and A l t h e s i n a p p l i c a t i o n s (9/32 and 8/30 neurons) compromised the a b i l i t y of neurons to f i r e r e p e t i t i v e l y in response to d i r e c t cu r r en t pu lse i n j e c t i o n through the r eco rd i ng e l e c t r o d e ( F i g . 6A-B). 3.1.2 E f f e c t s on Vm and membrane e l e c t r i c a l p r o p e r t i e s . A p p l i c a t i o n s of i s o f l u r a n e (1-2.5 MAC) hype rpo l a r i zed 24 of 34 c e l l s by 3-8 mV. This e f f e c t was s t a t i s t i c a l l y s i g n i f i c a n t only a t 2 and 2.5 MAC ( F i g . 7A). I s o f l u r ane adm in i s t r a t i on s at the h ighest dose evoked a mean h y p e r p o l a r i z a -t i o n (from c o n t r o l ) of 8.8 * 2.5% i n 5 neurons. The h y p e r p o l a r i z a t i o n e l i c i t e d w i th A l t h e s i n a p p l i c a t i o n s i n doses of more than 150 uM was s t a t i s t i c a l l y s i g n i f i c a n t ( F i g . 7B). S i g n i f i c a n t r educ t i on s i n i npu t r e s i s t a n c e (R^) were observed w i t h i s o f l u r a n e a p p l i c a t i o n s at 1.5, 2 and 2.5 MAC ( F i g . 7B). The mean decreases i n R.j a t these doses were 15.34%, 20.71% and 28.53%, r e s p e c t i v e l y . The e f f e c t of 1.5 MAC i s o f l u r a n e on R^  i s shown i n F i g . 8. A maximal r e d u c t i o n o f ~46% from c o n t r o l va lues was observed w i t h A l t h e s i n a p p l i c a t i o n s a t 1500 uM ( c f . F i g . 7D). Concomitant a p p l i c a t i o n of TTX (1.5 uM) and i s o f l u r -ane (1.5 MAC) or A l t h e s i n (100 nM) produced the same e f f e c t s on the V m and R.j i n 9 neurons as observed p r e v i o u s l y in the absence of TTX-blockade o f N a + - s p i k e genes i s . H. EL-BEHEIRY 44 F i g . 5 Anae s the t i c - i nduced inc reases i n the t h r e sho l d f o r sp ike genes i s . (A) us ing same cu r ren t i n t e n s i t i e s , i s o f l u r a n e (IFL 1 MAC) and A l t h e s i n (AL 100 uM) decreased the number of spikes evoked i n two sensor imotor neurons. Note the depress ion of the ampl i tude of the second sp ike and the po s t sp i ke AHP dur ing anae s the t i c a p p l i c a t i o n . was he ld cons tant throughout the exper iment. (B) no decrease i n r e p e t i t i v e f i r i n g occurred but a r educ t i on i n the AHPs was ev i den t dur ing A l t h e s i n a p p l i c a t i o n (50 nM). Complete recovery was observed a f t e r 9 min. Spikes are t runca ted due to frequency response of penwr i te r r eco rde r . V m d id not change dur ing the exper iment. IFL 1 MAC r e c o v e r i e s -MM JJ AL 100pM llOmV 50ms _ J B c o n t r o l m.. J L m AL 50 MM r e c o v e r y 1 0 m V UM _ n — i 75 m s 2nA oo 5 4^ cn H. EL-BEHEIRY 46 Control IFL1MAC Recovery AAAJ s J J 1nA Recovery 15 mV _ r F i g . 6 Anaes thet i c i n t e r f e r e n c e w i th the r e p e t i t i v e f i r i n g a b i l i t i e s o f n e o c o r t i c a l neurons. (A) i s o f l u r a n e (IFL 1 MAC) i n t e r f e r e d w i th r e p e t i t i v e f i r i n g e l i c i t e d by c u r r e n t pu l se i n j e c t i o n s . When the i n j e c t e d cu r r en t was inc reased ( lower t r a c e s ) , the a b i l i t y of the neuron to f i r e more than 1 l a r ge ampl i tude sp ike s t i l l was severe l y compromised. The AHPs a l s o were reduced. (B) A l t h e s i n (AL; 75 MM) depressed the r e p e t i t i v e f i r i n g a b i l i t i e s of an a n t e r i o r c i n g u l a t e neuron. V,^ i n A and B were c o n t r o l l e d a t the r e s t i n g l e v e l s ( ~ -74 mV). Sp ikes are t runca ted due to frequency response of penwr i t e r r e co rde r . 151 10 o a. w 01 a > » X 0.4 1 2 Isoflurane concentration IMACI 15 ion 'izat 10 ra •pol Hypei 5 B 10 100 Althesin concentration IjiMl 1000 1 2 3 10 100 1000 Isoflurane concentration IMACI Althesin concentration fMMI m i—i 73 -< F i g . 7 A l t e r a t i o n s i n the pass i ve membrane p rope r t i e s by a p p l i c a t i o n s of i s o f l u r a n e (A,B) and A l t h e s i n (C,D). Symbols ( • ) i n d i c a t e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e from ^ mean c o n t r o l va lues a t p<0.05. ^ H. EL-BEHEIRY 48 F i g . 8 E f f e c t s of i s o f l u r a n e (1.5 MAC f o r 6 min) on the i npu t r e s i s t a n c e of an a n t e r i o r c i n g u l a t e neuron. (A) responses to i n j e c t i o n s o f c u r r e n t pu l ses i n c o n t r o l , i s o f l u r a n e and recovery c o n d i t i o n s . Note t h a t i s o f l u r a n e a p p l i c a t i o n produced a s l i g h t decrease i n input r e s i s t a n c e . The t h re sho ld amount of c u r r en t f o r e l i c i t i n g one sp ike was i n c r ea sed . (B) the s l ope r e s i s t a n c e was decreased by ~28% dur ing i s o f l u r a n e a p p l i c a t i o n . Complete recovery was observed. H. EL-BEHEIRY 49 3.2 D i s cu s s i on The spontaneous a c t i v i t y of n e o c o r t i c a l neurons has been c o r r e l a t e d to the l e v e l of a c t i v a t i o n in the b r a i n (Webb 1983). This l e v e l i s o p t i m a l l y requ i red f o r r e c e i v i n g , ana l y z i ng and r eac t i n g t o i n t e r n a l and ex te rna l s t i m u l i ( K r n j e v i c 1974b; R ichards 1983). The observed depres s ion of spontaneous a c t i v i t y i n the s l i c e p repa ra t i on t ha t occur red w i thou t s i g n i f i -c an t , concomitant changes i n V m and R^, suggests t h a t at l e a s t some of the observed a c t i on s of i s o f l u r a n e and A l t h e s i n are at p r e s ynap t i c s i t e s . However, po s t s ynapt i c a c t i o n s are i n d i c a t e d because s i g n i f i c a n t i n c rea se s i n input conductance were observed wi th the anae s the t i c a p p l i c a t i o n s in the h igher dose range. When low doses of the anae s the t i c s were a p p l i e d , the f u l l extent of the changes in membrane conductance cou ld have been p a r t l y masked by anae s the t i c a c t i o n s t h a t decrease t r a n s m i t t e r r e l e a s e . A d d i t i o n of TTX, however, d i d not change the e f f e c t s of anae s t he t i c s on V m or R... Consequent ly, another p o s s i b i l i t y may be cons idered where the f a i l u r e to observe a change i n conductance would be a t t r i b u t a b l e t o anae s the t i c a c t i on s ( i n low doses) at d e n d r i t i c s i t e s d i s t a l to the r eco rd i ng e l e c t r o d e which was l i k e l y somatic i n l o c a t i o n . Th i s can e x p l a i n c e r t a i n i n c o n s i s t e n c i e s i n the l i t e r a t u r e about the e f f e c t s of anae s the t i c s on pa s s i ve membrane p r o p e r t i e s . For example, some i n v e s t i g a t i o n s have not revea led changes i n neuronal e x c i t a b i l i t i e s of CNS neurons (Zorychta and (Jfapek 1978; Sc hoi f i e l d 1980; Bosnjak e t a l . 1982 and Fu j iwa ra e t a l . 1988). Nonethe les s , profound reduc t i on s i n the e x c i t a b i l i t y of the po s t s ynap t i c membrane have been demonstrated dur ing anaes thes i a i n some c e n t r a l neurons ( N i c o l l and Madison 1982; Whitney and Glenn 1986; Berg-Johnson and Langmoen 1987; Takahashi and Takenosh i ta 1987) and i n p e r i p h e r a l sensory neurons w i thou t known synapt i c i nput s ( P u i l and Gimbarzevsky 1987). H. EL-BEHEIRY 50 The r e p e t i t i v e f i r i n g evoked by cu r ren t pu l se i n j e c t i o n s was depressed by the anae s t he t i c s , e s p e c i a l l y i s o f l u r a n e . Th i s e f f e c t cou ld be due to change i n N a + - i n a c t i v a t i o n i n the neuron (Hodgkin and Huxley 1952). A l o c a l a n a e s t h e t i c - l i k e e f f e c t on the Na + - channe l s seems u n l i k e l y because d im inu t i on s i n the ampl itude and shape of the evoked s i n g l e sp i kes were not observed i n most neurons de sp i te changes in the r a te of cu r r en t pu l se i n j e c -t i o n . In a d d i t i o n , the f i r s t sp i ke i n an evoked bu r s t was not a f f e c t e d by the anae s t he t i c a p p l i c a t i o n s ( F i g . 6 A - B ) . The compromised r e p e t i t i v e f i r i n g a b i l i t y can be a t t r i b u t e d to an a l t e r a t i o n i n the l e v e l of N a + - i n a c t i v a t i o n as a r e s u l t of anaes thet i c a t t enua t i on s of the AHPs ( c f . F i g s . 4B, 5 and 6 2+ and S e c t i o n VI I ) secondary to a depress ion of inward Ca - c u r r e n t s ( K r n j e v i c and P u i l 1988) which p a r t l y c o n t r i b u t e t o the a b i l i t y of n e o c o r t i -c a l neurons to f i r e sp ikes r e p e t i t i v e l y ( S ta f s t rom e t a l . 1985). 4 EFFECTS ON SYNAPTIC TRANSIENTS In t h i s s e r i e s of i n v e s t i g a t i o n s the e f f e c t s of i s o f l u r a n e (0.5-2.5 MAC) and A l t h e s i n (10 - 200 uM) a p p l i c a t i o n s were observed on 37 neurons. These neurons showed mean (iSEM) r e s t i n g p o t e n t i a l s of -74 * 4 mV and a mean R. of 65.9 * 3.65 Mn. They f i r e d r e p e t i t i v e sp ikes (<2 ms in du ra t i on ) of >70 mV i n ampl i tude w i t h overshoots of ~20 mV. The average du r a t i on of r e co rd i ng was 125 (±78) min. The e l e c t r o d e s were f i l l e d w i th K -acetate (2 M) and, on c e r t a i n o ther occas ions , KC1 (3 M) f o r the e x c i t a t o r y po s t s ynapt i c p o t e n t i a l s (EPSP) record ings o r C s 2 S 0 4 (3 M) f o r the dose/response s t ud i e s on i n h i b i t o r y po s t s ynapt i c p o t e n t i a l s ( IPSPs). 4.1 Re su l t s 4.1.1 Rest ing membrane p r o p e r t i e s . In t h i s s e r i e s , V m was monitored con t i nuou s l y on a pen recorder whereas R. was monitored by i n j e c t i o n o f H. EL-BEHEIRY 51 h y p e r p o l a r i z i n g pulses in between two succes s i ve s t i m u l a t i o n s or by i n t e r -r up t i n g the rhythmic s t i m u l a t i o n (0.2 Hz) j u s t be fo re , a t the end, and a f t e r ~12 min of an anaes the t i c a p p l i c a t i o n . An i s o f l u r a n e - i n d u c e d h y p e r p o l a r i z a -t i o n was observed only a t doses >1.5 MAC. A p p l i c a t i o n a t the h i ghe s t dose (2.5 MAC) hype rpo la r i zed 3 neurons by 3-5 mV. The h y p e r p o l a r i z a t i o n s e l i c i t e d by A l t h e s i n were not s t a t i s t i c a l l y s i g n i f i c a n t even a t the 200 nM dose where 4 neurons were hype rpo l a r i zed by ~4 mV. S t a t i s t i c a l l y s i g n i f i c a n t changes i n R^  were observed dur ing i s o f l u r a n e a p p l i c a t i o n s a t 1.5, 2 and 2.5 MAC. In 3 neurons, the maximum mean decrease from c o n t r o l va lues was 25% a f t e r 8 min of a p p l i c a t i o n (2.5 MAC). No s i g n i f i c a n t changes i n _R. were observed dur ing A l t h e s i n a p p l i c a t i o n s at a l l doses (10-200 ^M). 4.1.2 Dose-response depress ion of e x c i t a t o r y pos t synapt i c p o t e n t i a l s  (EPSPs). Foca l s t i m u l a t i o n i n the immediate subp ia l reg ion of the n e o c o r t i -c a l s l i c e produced EPSPs in the pyramidal c e l l s l o ca ted at the j u n c t i o n of the middle and lower t h i r d po r t i on s of the c o r o n a l l y sec t ioned s l i c e . By us ing cons tant j u s t - t h r e s h o l d s t i m u l i (4-15 V; 0.1 ms), EPSPs w i thout a c t i o n p o t e n t i a l s were i n i t i a t e d i n ~50% of these s t i m u l a t i o n exper iments (a t 0.2 Hz) . When necessary ( i . e . , when an anae s the t i c induced a change i n V ) the r e s t i n g p o t e n t i a l was he ld cons tant a t the i n i t i a l r e s t i n g l e v e l by cont inuous DC-current i n j e c t i o n throughout the exper imenta l p e r i o d . The mean ampl itude (±SEM) of the c o n t r o l EPSPs was 18.5 (±0.55) mV w i th an average du r a t i on of 210 (±30) ms ( c f . A v o l i and O l i v i e r 1989). The mean r a t e of r i s e was 0.25 (±0.02) mV/ms whereas the ra te of decay was 0.07 (±0.01) mV/ms. A p p l i c a t i o n of i s o f l u r a n e (1-2.5 MAC) produced a dose-dependent r e v e r s i b l e depress ion i n the ampl i tude of the EPSPs (16/16 neurons; F i g . 9A). A t 1 and 2 MAC the mean depress ions from the c o n t r o l va lues were 52.56 and 80.25% r e s p e c t i v e l y ( F i g . 10). The r a te s of r i s e of H. EL-BEHEIRY 52 F i g . 9 EPSP-depress ion induced by i s o f l u r a n e and A l t h e s i n . (A) i s o f l u r a n e ( I FL ) was app l i ed to two sensor imotor neurons f o r 10 min i n doses of 1 and 2 MAC. The superimposed t r a c e s show almost complete depress ion a t 2 MAC. Recovery was ev ident a f t e r 12 min. (B) A l t h e s i n 75 and 200 was app l i ed t o two a n t e r i o r c i n g u l a t e neurons f o r 8 min. P a r t i a l recovery was obta ined a f t e r 15 min i n the second neuron ( lower t r a c e ) . H. EL-BEHEIRY 53 150 ms IFL 1 .5 MAC B 100-r 0.4 0.5 1.0 2.0 0.4 0.5 1.0 2.0 Isoflurane (MAC) Isoflurane (MAC) F i g . 10 Dose-dependent i s o f l u r a n e (0 .5-2.5 MAC), sensor imotor neuron du r i ng c o n t r o l and i s o f l u r a n e ( IFL; 1.5 using the same s t imu lu s s t r eng th and du r a t i o n . Traces obta ined over a pe r i od of 2 min be fo re , and at the end a p p l i c a t i o n ( frequency of s t i m u l a t i o n was 0.2 Hz ) . (B) r e l a t i o n s h i p s f o r the percent depress ion of amp l i tudes , r a t e s of r i s e and ra te s of decay of the averaged EPSPs. The sample s i z e f o r each dose was >4. S t a t i s t i c a l s i g n i f i c a n c e (p<0.05) from c o n t r o l va lues i s i n d i c a t e d by T*). Note tha t depres s ion of the r a t e of r i s e was s i g n i f i c a n t a t 2.5 MAC. E r r o r bars represent s tandard d e v i a t i o n s . supres s ion of the ampl itude and r a t e of decay by (A) computer-averaged t r a c e s of EPSPs evoked i n a MAC) pe r f u s i on averaged were of i s o f l u r a n e dose-response H. EL-BEHEIRY 54 the EPSP were not s i g n i f i c a n t l y a f f e c t e d except at concent ra t i on s >2 MAC whereas the r a te s of decay were reduced i n a dose-dependent manner. A t 0.5 and 1.5 MAC i s o f l u r a n e , f o r example, the mean percent depress ions from c o n t r o l c o n d i t i o n s were 18.8 (±3.67, S.E.M.) mV/ms and 47.74 (±1.74) mV/ms r e s p e c t i v e l y ( F i g . 10) . The dose-dependent e f f e c t s of A l t h e s i n occurred w i t h a t ime course s i m i l a r to t ha t of the i s o f l u r a n e e f f e c t s i n a l l the neurons (n = 13). Pe r fu s i on of the v e h i c l e , cremophor EL (0.002 -0.016% v / v ) , had no e f f e c t on EPSPs, or on the membrane conductance (n = 4 neurons) . The E C 5 Q f o r the depress ion of the EPSP ampl itude by A l t h e s i n was s l i g h t l y above 50 uM ( F i g . 11) and complete suppress ion was observed at 200 uM. However, A l t h e s i n was l e s s potent than i s o f l u r a n e i n depress ing the r a te s of decay; the mean maximum percent depres s ion was 77.88 (±2.9) w i th c oncen t r a t i on s as high as 200 uM ( F i g . 11). 4.1.3 EPSP a t t enua t i on i n the presence of IPSP-b lockade. Because the observed p ro l onga t i on of a l l o c o r t i c a l IPSPs are presumed to c o n t r i b u t e to the depres s ion of the EPSPs produced by anae s the t i c agents ( S c h o l f i e l d 1980), b i c u c u l l i n e (50 uM) was used to pha rmaco log i ca l l y i s o l a t e the EPSPs f o r a more exact i s o l a t i o n of the e f f e c t s of i s o f l u r a n e and A l t h e s i n . Using K -aceta te or C s 2 S 0 4 e l e c t r ode s ( c f . F i g . 14C), blockade of the IPSPs was observed w i t h i n 5 min of b i c u c u l l i n e pe r f u s i o n a t a f l ow r a t e of 2 ml/min. No d e p o l a r i z a t i o n or i nc rease in R^  was observed dur ing a p p l i c a t i o n of the c onvu l s an t , p o s s i b l y due t o the preponderance of t o n i c e x c i t a t o r y i nput s imping ing on these neurons ( c f . Matsumura e t a l . 1988; Buzsaki 1984). Under such c o n d i t i o n s , the s t imu lu s s t reng th t ha t was r equ i r ed to e l i c i t the c o n t r o l , j u s t - t h r e s h o l d EPSPs i n i t i a t e d on ly e p i l e p t i f o r m burs t s t h a t u s u a l l y were a s soc i a ted w i t h 2-3 a c t i o n p o t e n t i a l s ( F i g . 12A). I s o f l u r ane (1.5 MAC) and A l t h e s i n (75 nM) a p p l i e d concomi tant l y w i th b i c u c u l l i n e H. EL-BEHEIRY 55 A o -j 1 1 1 1 — -| "—i 1 1 1 — 10 20 50 100 200 10 20 50 100 200 Althesin (/xM) Althesin (/xM) F i g . 11 A l t h e s i n - i n d u c e d dose-dependent depress ion of the ampl itudes and r a te s of decay of n e o c o r t i c a l evoked EPSPs. (A) computer-averaged t r a ce s evoked i n an a n t e r i o r c i n g u l a t e neuron are shown before and dur ing A l t h e s i n a p p l i c a t i o n (AL; 50 LIM). Each averaged- t race represent s a t l e a s t 15 i n d i v i d u a l t r a c e s . (B) ampl itudes and r a te s of decay were s i g n i f i c a n t l y depressed i n a dose-dependent manner, whereas the ra te s of r i s e showed s t a t i s t i c a l l y s i g n i f i c a n t suppress ion on ly at the h ighes t dose (200 u.M). Symbols (*) i n d i c a t e s t a t i s t i c a l s i g n i f i c a n c e (p<0.05) from c o n t r o l . Sample s i z e s were 2.4 except at 15 and 200 LIM (n = 3 in each) . H. EL-BEHEIRY 56 F i g . 12 Ep i l ep togen i c a c t i v i t y and EPSP suppress ion by anae s the t i c a p p l i c a t i o n s i n the presence of GABAergic blockade i n n e o c o r t i c a l neurons. (A) A p p l i c a t i o n of b i c u c u l l i n e (BIC; 50 MM) i n i t i a t e d e p i l e p t i c a c t i v i t y i n a neuron t h a t was responding to the same s t imu lus w i th an EPSP. (B-C) e p i l e p t i c bur s t s induced by b i c u c u l l i n e in two neurons were suppressed by the a p p l i c a t i o n of i s o f l u r a n e and A l t h e s i n ( IFL, 1.5 MAC, 15 min and AL, 75 nM, 12 min r e s p e c t i v e l y ) . In (B) the time course of bur s t suppress ion i s shown (middle t r a c e , 5 min and lower t r a c e , 12 min) . (D) i n low C a 2 + media an EPSP cou ld be evoked dur ing the concomitant a p p l i c a t i o n of b i c u c u l l i n e . Under such c o n d i t i o n s , a concomitant anae s the t i c a p p l i c a t i o n (~10 min) at tenuated the EPSPs i n a s i m i l a r manner to tha t observed i n the absence of b i c u c u l l i n e ( c f . F i g . 9 ) . H. EL-BEHEIRY 58 depressed the ampl itudes of the e p i l e p t i f o r m b u r s t s , unmasking small EPSPs ( F i g . 12B-C). In order to e l i m i n a t e the a c t i o n p o t e n t i a l s and e p i l e p t i f o r m 2+ a c t i v i t y , the e x t r a c e l l u l a r Ca concen t r a t i on was decreased to 1 mM and 2+ the [Mg ] was i nc reased by an e q u i v a l e n t amount. Because of t h i s procedure, EPSPs subthresho ld to spike genesis cou ld be evoked. A f t e r the a p p l i c a t i o n of i s o f l u r a n e and A l t h e s i n (1.75 MAC i n 4 neurons and 75 ^M i n 4 a d d i t i o n a l neurons, r e s p e c t i v e l y ) , the EPSPs were depressed i n a manner s i m i l a r t o t h a t observed i n the absence of b i c u c u l l i n e ( F i g . 12D). 4.1.4 E f f e c t s on o r t hod rom i ca l l y and i n t r a c e l l u l a r ^ evoked sp i ke s . Supramaximal s t i m u l a t i o n c o n s i s t e n t l y evoked sp i ke s tha t were blocked by anae s the t i c pe r fu s i on ( i s o f l u r a n e at 1.5 MAC to 3 neurons and A l t h e s i n i n 50 uM doses to a d d i t i o n a l 3 neurons) f o r 12 min a t f l ow r a te of 2 ml/min ( F i g . 13, A l - B l ) . Recovery was ev i den t a f t e r ~15 min on r e t u r n i n g to the c o n t r o l s o l u t i o n . On i n t e r r u p t i n g the subp ia l s t i m u l a t i o n , more than two sp ikes were evoked in these neurons w i th i n j e c t i o n s of d e p o l a r i z i n g cu r r en t pu l s e s . The same c u r r e n t i n j e c t i o n i n t e n s i t i e s used dur ing the anaes the t i c a p p l i c a t i o n s revea led the compromised a b i l i t i e s of the neurons to f i r e the same number of sp i kes as i n c o n t r o l c o n d i t i o n s ( F i g . 13A2-B2). 4.1.5 Dose-response r e l a t i o n s h i p f o r IPSP suppress ion dur ing  Cs -b lockade o f the K-conductances. The use of i n t r a c e l l u l a r C s 2 S 0 4 e l e c t r o d e s was a s s o c i a t e d a f t e r about 15 min of impalement (n = 17 neurons), w i t h an i nput r e s i s t a n c e which inc reased from a mean c o n t r o l va lue of 55.75 *3.25MQ to a mean of 74.57 (*4.25) Mn , an average d e p o l a r i z a t i o n o f ~11 mV and a markedly prolonged sp i ke du r a t i on ( F i g . 14A). A f t e r 20 min of C s - a p p l i c a t i o n the po s t sp i ke a f t e r h y p e r p o l a r i z a t i o n was complete ly abo l i s hed ( F i g . 14B). A p p l i c a t i o n of b i c u c u l l i n e a t t h i s po i n t b locked the IPSPs (4 neurons; F i g . 14C). Thus the use of the C s 9 SO . - con ta i n i n g e l e c t r o d e s H. EL-BEHEIRY 59 B2 I AL 50M  F i g . 13 Anaes the t i c i n t e r f e r e n c e w i th the s y n a p t i c a l l y - i n d u c e d sp ikes and d i r e c t l y - e v o k e d a c t i o n p o t e n t i a l s by i n t r a c e l l u l a r c u r r e n t pu lse i n j e c t i o n s . S y n a p t i c a l l y - i n d u c e d (A l ) and d i r e c t l y - e v o k e d sp ikes (A2) were depressed by i s o f l u r a n e ( IFL ; 1 MAC) i n a sensor imotor neuron. (B l and B2) the same e f f e c t was produced by A l t h e s i n (AL; 50 LIM) i n another sensor imotor neuron. C a l i b r a t i o n ba r s : 120 ms ( h o r i z o n t a l ) and 10 mV ( v e r t i c a l ) i n A l and B l , 100 ms and 40 mV or 1 nA i n A2 and B2. H. EL-BEHEIRY 60 I n t e r n a l C s + - a p p l i c a t i o n o 111.. -70" 3 min u 15 min 50 ms "J20mV B 2 min 20 min o.3s 10 mV D 160ms BIC 5mV F i g . 14 I n te rna l C s + - a p p l i c a t i o n i n 4 n e o c o r t i c a l neurons. (A) C s + leakage from an i n t r a c e l l u l a r m i c r o p i p e t t e caused p ro l onga t i on of the a c t i o n p o t e n t i a l s , p a r t i a l b lockade of the AHP and a small l o c a l a n a e s t h e t i c - l i k e e f f e c t i . e . , a r educ t i on i n the r a t e s of r i s e and ampl i tudes of the sp i kes a f t e r 15 min of impalement. (B) i n another a n t e r i o r c i n g u l a t e neuron complete blockade of the AHP was ev ident a f t e r 20 min of p e n e t r a t i o n . In (A) and (B ) , the membrane p o t e n t i a l (V m ) was kept cons tant a t the r e s t i n g l e v e l by DC-current i n j e c t i o n . In both c e l l s V m was d e p o l a r i z e d by 13 and 15 mV r e s p e c t i v e l y a f t e r ~25 min. (C) complete suppress ion of an IPSP a f t e r b i c u c u l l i n e a p p l i c a t i o n (BIC; 50 uM; 5 min) i n a neuron impaled by a CS2SO4 e l e c t r o d e . (D) t ime course of blockade of the IPSPs i n a sensor imotor neuron penet rated w i t h a CS2SO4 m i c r o p i p e t t e . Lower and middle t r a ce s were obta ined a f t e r 1 and 3 min of b i c u c u l l i n e (50 uM) p e r f u s i o n . In (C) and (D). IPSPs were evoked a f t e r complete a t t e n u a t i o n of the AHP and at ta inment o f a steady s t a t e of V m (-48 mV from a r e s t i n g l e v e l -66 mV). H. EL-BEHEIRY 6 1 removed any " c on t am ina t i on " of the IPSPs by K-mediated l a t e h y p e r p o l a r i z i n g p o t e n t i a l s . In a d d i t i o n , the Cs- induced d e p o l a r i z a t i o n i nc rea sed the amplitude of the evoked IPSPs and rendered them more amenable f o r ex tens i ve examinat ion of d rug - a c t i on s . The mean ampl itude of the IPSPs was 5 . 2 mV ( ± 0 . 3 5 ) whereas the mean du ra t i on and rate of decay were 4 1 0 ( ^ 4 2 ) ms and 0 . 0 1 3 ( ± 0 . 0 0 7 ) mV/ms r e s p e c t i v e l y ( c f . Howe e t a l . 1 9 8 7 ; Su tor and H a b l i t z 1 9 8 9 ) . The a p p l i c a t i o n of i s o f l u r a n e or A l t h e s i n c o n s i s t e n t l y depressed the IPSPs i n a l l neurons (n = 1 7 ) ; t h i s e f f e c t was dose-dependent, r e v e r s i b l e and r ep roduc i b l e on repeated a p p l i c a t i o n s to the same neuron. I s o f l u r ane ( 1 . 5 MAC) and A l t h e s i n ( 7 5 uM) depressed the ampl i tudes of IPSPs by ~ 5 4 and 55% r e s p e c t i v e l y ( F i g s . 1 5 and 1 6 ) . The e f f e c t s were on ly ev i den t a f t e r prolonged a d m i n i s t r a t i o n ( 1 2 - 1 5 min) in the case of both agents . Recovery u s u a l l y was observed a f t e r 2 0 min. The E C ^ Q f o r the a t t e n u a t i o n of the IPSPs was g r e a t e r than E C 5 Q f o r depress ion of the EPSPs i n the case of both agents. A l though a dose-dependent s lowing of the IPSP decay was ev i den t , the dose-response curves f o r both agents had a lower s lope values than t h a t f o r the EPSPs. The maximum depress ions of the r a t e of decay from the c o n t r o l values were ~ 5 4 % f o r 2 . 5 MAC i s o f l u r a n e and 4 5 % f o r 1 0 0 uM A l t h e s i n . S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were observed i n the h igher dose range ( F i g . 1 6 ) . Moreover, complete recovery of the ra te of decay was observed i n ~ 7 0 % of neurons. 4 . 2 D i s cu s s i on The e a r l y s t ud i e s by Ecc l e s ( 1 9 4 6 ) , Brookes and E c c l e s ( 1 9 4 7 ) and Larrabee and Posternak ( 1 9 5 2 ) demonstrated t h a t e x c i t a t o r y s ynap t i c t r a n s -mi s s i on was reduced by a d m i n i s t r a t i o n of va r i ou s genera l anae s the t i c s i n c l u d i n g c h l o r o f o r m , d i e t h y l e t h e r and b a r b i t u r a t e s . L a t e r s t ud i e s by Somjen control IFL 1.5 MAC recovery 250ms B controls AL 35,,M recoveries 5mV AL 75/iM F i g . 15 IPSP -a t tenuat ion by i s o f l u r a n e and A l t h e s i n a f t e r K + -conductance blockade by i n t r a c e l l u l a r a p p l i c a t i o n of C s + . (A) i s o f l u r a n e (1.5 MAC; 12 min a p p l i c a t i o n ) suppressed the IPSPs by ~60% . (B) A l t h e s i n (35 and 75 L.M; 12 min a p p l i c a t i o n ) attenuated of the IPSPs evoked i n sensor imotor and a n t e r i o r c i n g u l a t e neurons r e s p e c t i v e l y . In (A) and (B) the superimposed t r a ce s were at l e a s t 6 in each c o n d i t i o n . H. EL-BEHEIRY 63 IFL1MAC \ B A L ^ 5 / i M C 100 -r BO - -o *W 80 + o a* & « 20--O Amplitude A Rate of decaj 0.5 1.0 2.0 Isoflurane (MAC) OAmplitude ARate of decay -I > ' r — 10.0 20.0 50.0 100.0 Althesin (JJM) neocortical neurons + . (A F i g . 16 Dose-dependent interference with the IPSPs in with concomitant blockade of K +-conductances by i n t r a c e l l u l a r Cs and B) computer-averaged traces i n two neurons i n which isof lurane and Althes in (IFL, 1 MAC; AL, 75 uM) were appl ied. (C and D) dose-response curves fo r isof lurane and Al thes in showing suppression of the amplitudes and rates of decay by at least 3 anaesthetic appl icat ions for each dose. H. EL-BEHEIRY 64 and G i l l (1963) and Somjen (1963) conf i rmed t h a t d i e t h y l e t h e r and b a r b i t u r -a tes depressed the EPSPs i n motoneurons. Hence c l e a r ev idence was obta ined f o r d i r e c t ac t i on s of anae s t he t i c s on c e n t r a l s ynapt i c t r a n s m i s s i o n . Subsequent ly, halothane and b a r b i t u r a t e s were shown to depress e x c i t a t o r y s ynap t i c t r an sm i s s i on i n the cuneate nucleus (Gal indo 1969). A t the suprasp ina l l e v e l s , a wide v a r i e t y of anaes the t i c s has now been found to depress e x c i t a t o r y s ynap t i c t r an sm i s s i on p a r t i c u l a r l y i n the hippocampal fo rmat ion (Berg-Johnson and Langmoen 1986a,b and 1987; Mac lve r and Roth 1988; Miu and P u i l 1989) and o l f a c t o r y cor tex (R ichards 1973; R ichards e t a l . , 1975). However, N i c o l l (1972) has shown tha t the unusual dendro-d e n d r i t i c e x c i t a t o r y synapses of the o l f a c t o r y bulb are r e l a t i v e l y r e s i s t a n t to the depres s i ve a c t i o n s of general anae s the t i c s . Moreover, Fu j iwa ra and h i s a s s oc i a te s (1988) a l s o found no e f f e c t s of i s o f l u r a n e , halothane o r en f l u rane on evoked EPSPs i n the hippocampal s l i c e i n v i t r o . There i s no c l e a r ev idence at p re sent t i m e , t h a t any general a nae s t he t i c a c t u a l l y i nc rea se s the e f f i c a c y of e x c i t a t o r y synapses. Such a mechanism might be expected to p r e v a i l du r ing the e a r l y e x c i t a t o r y phase of the anae s t he t i c s t a t e o r , g iven e x c i t a t o r y s ynapt i c i nput s to i n h i b i t o r y i n t e rneu ron s , cou ld c o n t r i b u t e to the enhanced i n h i b i t o r y processes dur ing anae s t he t i c admin i s -t r a t i o n (see below; c f . M o r r i s 1978). The a c t i o n s of genera l anae s the t i c s on i n h i b i t o r y s ynapt i c t r an sm i s s i on are more complex than on those at e x c i t a t o r y synapses. E c c l e s and a s s o c i a t e s (1963) repor ted t h a t p re synap t i c i n h i b i t i o n was enhanced by b a r b i t u r a t e or c h l o r a l o s e a d m i n i s t r a t i o n but depressed by d i e t h y l e t h e r and c h l o r a l hydrate a p p l i c a t i o n . Larson and Major (1970) reported t ha t hexobarbitone a p p l i c a t i o n inc reased the du ra t i on of IPSPs i n the sp ina l cord i n i n v i v o f e l i n e p r epa r a t i on s . L a t e r , N i c o l l and h i s a s s oc i a te s (1975) a l s o observed a H. EL-BEHEIRY 65 b a r b i t u r a t e - i n d u c e d p r o l o n g a t i o n of i n h i b i t o r y p o t e n t i a l s i n the hippocampus of i n v i vo f e l i n e p r e p a r a t i o n s . More r e c e n t l y , i n c rea se s i n the spontaneous IPSCs (Gage and Robertson 1985) and the evoked IPSPs (Miu and P u i l 1989) have been observed i n hippocampal i n v i t r o s l i c e p r epa ra t i on s . In i n v i t r o s t ud i e s of the o l f a c t o r y bulb and cor tex (Sc hoi f i e l d 1978, 1980) which were i n i t i a t e d by N i c o l l (1972), a d m i n i s t r a t i o n of b a r b i t u r a t e s , ha lothane, a lphaxalone and ketamine inc reased the du ra t i on of po s t s ynap t i c i n h i b i t i o n . In the s p i n a l c o r d , Weakly (1968) d id not observe any s p e c i f i c i nc rea se i n the d i r e c t i n h i b i t i o n of motoneurons i n v i vo whereas depres s ion of IPSPs was ev ident i n i n v i t r o s l i c e p repa ra t i on s of the newborn r a t s p i n a l cord (Takahashi and Takenosh i ta 1987). S y n a p t i c a l l y evoked i n h i b i t o r y p o t e n t i a l s recorded i n the CA1 pyramidal c e l l s of the hippocampus are c o n s i s t e n t l y attenuated by c l i n i c a l l y used i n h a l a t i o n a l anae s the t i c s (Yoshimura e t a l . 1985; Fu j iwa ra e t a l . 1988). M o d i f i c a t i o n of the synapt i c t r a n s i e n t s dur ing anaes thes ia have not been i n v e s t i g a t e d e x t e n s i v e l y i n the neocortex which i s an area t h a t i s very vu lne rab le to anae s the t i c a c t i o n s ( c f . I n t r o d u c t i o n ) . In a d d i t i o n , e x p e r i -mental procedures to i s o l a t e the conductances a s s oc i a t ed w i th the EPSPs o r IPSPs from other " c on tam ina t i n g " conductances have not been implemented i n the prev ious i n v e s t i g a t i o n s . This may have r e s u l t e d in some of the apparent c o n t r a d i c t i o n s a r i s i n g from the d i f f e r e n t exper imenta l p r epa ra t i on s used by prev ious i n v e s t i g a t o r s and a l s o may have prov ided some d i f f i c u l t y i n i n t e r -p r e t i n g the " t r u e e f f e c t s " on these t r a n s i e n t s . 4.2.1 Anae s the t i c depres s ion of EPSPs. A s t r i k i n g f ea tu re of the anaes the t i c e f f e c t s on EPSPs i s the dose-dependent depres s ion i n t h e i r ampl itudes w i t h E C 5 0 s of 1 MAC and ~50 LIM f o r the r e s p e c t i v e depress ions by i s o f l u r a n e and A l t h e s i n ; these concen t ra t i on s are s i m i l a r t o those t ha t H. EL-BEHEIRY 66 have been determined i n the b r a i n of anae s the t i zed animals (Smith e t a l 1974; M i l l s et a l . 1987). An i n t e r e s t i n g e f f e c t which was observed i n the present i n v e s t i g a t i o n s was the dose-dependent s lowing in the ra te of decay of the s ynapt i c t r a n s i e n t s . Quastel and L i nde r (1975) and Gage and a s s o c i a t e s (1975 and 1979) have repor ted s i m i l a r e f f e c t s of c e r t a i n a l i p h a t i c a l c o h o l s on the m in ia tu re endplate c u r r e n t s a t the neuromuscular j u n c t i o n . However, comparable f i n d i n g s have not been p r e v i o u s l y observed i n neurons of the CNS ( c f . Kullman et a l 1989). The anae s the t i c depress ion of EPSP ampl i tude i n neocortex cou ld be due to p re s ynap t i c or pos t synapt i c a c t i o n s . P re synapt i c mechanisms i n c l ude depres s ion of impulse conduct ion or nerve te rm ina l e x c i t a b i l i t y (Quastel and S a i n t 1986; Berg-Johnsen and Langmoen 1986b; S a i n t et a l . 1986; Bu t te rwor th et a l 1989) and, depress ion of t r a n s m i t t e r r e l ea se e i t h e r by i n t e r f e r i n g wi th s yn thes i s of t r a n s m i t t e r and/or i t s m o b i l i z a t i o n , thereby i n h i b i t i n g the number of re leased quanta per impulse or decreas ing the quantal content (Cheng and Brunner 1978; Zorychta and Capek 1978; Lodge and An i s 1984; Johnson 1985; Bosnjak et a l . 1988; Kullman e t a l 1989). P o s t s y n a p t i c a l l y , an i nc rease i n membrane conductance (N i c o l and Madison 1982) or a decrease i n the recepto r s e n s i t i v i t y to a t r a n s m i t t e r (Anis et a l . 1983; Sawada and Yamamoto 1985) can l e ad t o a t t e n u a t i o n of EPSPs. The observed suppress ion of the EPSP ampl i tudes cou l d be exp la ined by a summation of p re - and pos t synapt i c depressant a c t i on s of the anae s the t i c agents. The depress ion of the C a - a c t i v a t e d K-conductance t ha t was " i s o l a t e d " i n n e o c o r t i c a l neurons ( c f . S e c t i o n 6) p rov ide s i n d i r e c t evidence t h a t a Ca-conductance may be depressed i n the p re synap t i c t e r m i n a l . For 2+ example, an a t tenuated Ca i n f l u x would decrease the number o f quanta re l ea sed per s t imu lu s . This p o s s i b i l i t y i s supported by the observat ions of H. EL-BEHEIRY 67 K r n j e v i c and P u i l (1988) who observed a depress ion of c e r t a i n v o l t a g e -dependent Ca cu r r en t s i n vo l tage-c lamped hippocampal CA1 neurons dur ing halothane a p p l i c a t i o n ( a l s o c f . Morgan and Bryant 1977 and N i s h i and Oyama 1983). Zorychta and c o l l a b o r a t o r s (1975; Zorychta and Capek 1978) repor ted t h a t the decreased number of quanta re l ea sed dur ing halothane and d i e t h y l -e t he r anaes thes i a was the mechanism of suppress ion of monosynaptic EPSPs i n the sp i na l cord of c a t s . The p ro l onga t i on of the decay w i t h a concomitant depres s ion of the ampl i tude of the EPSPs cou ld be due t o : (a) a p r opo r t i ona l supress ion of the uptake mechanisms ( c f . P u i l , 1981; M inch in 1981) w i th a reduc t i on i n the number of r ecepto r s a c t i v a t e d ; (b) a p ro l onga t i on of the membrane time cons tant due to an increased i npu t capac i tance at membrane s i t e s d i s t a n t from the somatic reco rd ing e l e c t r o d e ( c f . P u i l and Gimbarzevsky 1987); (c) an inc rease in the mean open t ime of the r ecep to r - ope ra ted channe l s med iat ing the EPSPs, concomitant w i t h a decrease i n the abso lu te number of r ecepto r s a c t i v a t e d or d) the s imultaneous depress ion of the IPSPs dur ing the anae s the t i c a p p l i c a t i o n . The r e s u l t s of the present s t ud i e s are a t va r i ance w i t h the observat ions repor ted i n o l f a c t o r y co r tex and hippocampal f o rmat i on . S c h o l f i e l d (1980) a t t r i b u t e d the depress ion of the EPSPs i n o l f a c t o r y co r tex t o a shunt ing e f f e c t produced by the anae s the t i c p r o l onga t i on of the IPSPs. In the p resent i n v e s t i g a t i o n s b i c u c u l l i n e (50 uM) was a p p l i e d t o n e o c o r t i c a l neurons before anae s the t i c a p p l i c a t i o n and the IPSPs were a d d i t i o n a l l y monitored i n o rder t o e l i m i n a t e t h i s p o s s i b i l i t y . Once blockade of IPSP was observed, i s o f l u r -ane o r A l t h e s i n was per fused s imul taneous ly w i th b i c u c u l l i n e . C l i n i c a l l y H. EL-BEHEIRY 68 r e l e v a n t doses of both agents not on ly depressed the EPSPs but a l s o were e f f e c t i v e i n i n h i b i t i n g the e p i l e p t i f o r m neuronal d i scharges i n i t i a t e d by the GABA^-antagonist ( c f . F i g . 12) . In the hippocampus, EPSPs recorded i n the CA1 neurons were not a f f e c t e d by the a p p l i c a t i o n s of i s o f l u r a n e , en f l u r ane or halothane (Yoshimura e t a l . 1985; Fu j iwa ra e t a l . 1988). Accord ing to the l a t t e r r e p o r t s , a n o n - a i r - t i g h t gas chamber was used and the anae s the t i c s were a p p l i e d on l y by bath pe r fu s i on and were not i n t roduced i n t o the atmosphere above the s l i c e . In a d d i t i o n , the v o l a t i l e agents were not bubbled i n t o the pe r fu sa te f o r a s u i t a b l e e q u i l i b r a t i o n pe r i od but were added and "shaken v i g o r o u s l y " i n a stock s o l u t i o n before d i l u t i o n and a p p l i c a t i o n in the chamber. There fo re , leakage of anae s the t i c vapour cou ld have occurred dur ing such a p p l i c a t i o n s and cou ld have l ead to a decrease i n the concent ra t i on s of the agents at the e f f e c t o r s i t e s ( c f . B a z i l e t a l . 1987). 4.2.2 Anaes the t i c a c t i o n s on the IPSPs. The use o f CsgSO^ e l e c t r ode s i n these experiments p rov ided two main advantages: (a) lower t i p r e s i s t a n c e s than i n the case of K -acetate f i l l e d m i c ro -p i p e t t e s and (b) blockade of K + - conductances t h a t cou ld have contaminated endogenous a c t i v i t y mediated by GABA evoked C l " i n f l u x . The dose-dependent depres s ion of the IPSP ampl i tudes together w i t h the decrease i n the r a t e of decay suggests a common p r e - and po s t s ynap t i c mechanism f o r the depress ion of the IPSPs and the EPSPs. Nonethe less , i f i n h i b i t o r y in terneurons are a c t i v a t e d by e x c i t a t o r y synapses, the anae s the t i c - i nduced depres s ion of EPSPs may l ead t o a concomitant a t t e n u a t i o n of the IPSPs. H. EL-BEHEIRY 69 Takahashi and Takenosh i ta (1987) a l so observed an a t t e n u a t i o n of the evoked EPSPs and IPSPs recorded from motoneurons i n i n v i t r o s l i c e p repara -t i o n s . However, t h e i r r e s u l t s and those of the p resent i n v e s t i g a t i o n s c o n t r a d i c t the anae s the t i c p o t e n t i a t i o n of the spontaneous and evoked IPSCs and IPSPs repor ted f o r o l f a c t o r y co r tex and hippocampal neurons ( S c h o l f i e l d 1980; Gage and Robertson 1985; Ha r r i s on e t a l . 1987). De sp i te the fundamental d i f f e r e n c e s between the types of t o n i c s ynapt i c i nput s i n the n e o c o r t i c a l (Matsumura e t a l . 1988), hippocampal CA1 (Buzsak i 1984) and o l f a c t o r y c o r t i c a l neurons ( S c h o l f i e l d 1978), the d i s c r epan t f i n d i n g s cou ld have a r i s e n from the a c t i v a t i o n of K + -conductances t h a t " con tamina te " the IPSPs i . e . , i n the o l f a c t o r y c o r t i c a l and hippocampal neurons where i n t e r n a l C s + had not been a p p l i e d . Another p o s s i b i l i t y i s tha t the low temperature (20-25°C) used dur ing the experiments i n the a l l o c o r t e x ( i . e . , o l f a c t o r y co r t ex and hippocampus) would tend to slow down the k i n e t i c s of a c t i v a t e d i o n i c channels and may i nc rease t h e i r mean open-t ime. In the o l f a c t o r y c o r t e x , an anaes thet i c depress ion of the EPSPs a l s o was observed. Hence, the enhancement of the IPSPs cou ld be a t t r i b u t e d to a suppres ion of EPSPs which cou ld have unmasked the f u l l ex tent of the i n h i b i t o r y i o n i c c u r r e n t s . In the case of the a lphaxa lone a p p l i c a t i o n s to c u l t u r e d hippocampal neurons (Har r i son e t a l . 1987), the doses which were used (0.1-10 uM) were s i x t imes lower than those t h a t occur i n the b r a i n grey matter of anae s the t i z ed r a t s (Smith et a l . 1974). A t such low concen t ra t i on s i n the i n v i vo s i t u a t i o n a lphaxalone may act as a s e d a t i v e , e . g . , by p ro long ing the IPSPs, but would l i k e l y not induce anae s the s i a , p a r t i c u l a r l y s i n ce p rev ious s t u d i e s w i t h a lphaxalone have revea led a depress ion of GABAergic responses i n midbra in neurons of ve r t eb ra te s ( C u l l e n and M a r t i n 1982). H. EL-BEHEIRY 70 5 ANAESTHETIC INDUCED ALTERATIONS IN NEURONAL RESPONSIVENESS  TO ACTIVATING TRANSMITTERS AND RELATED SUBSTANCES The mean (±SD) r e s t i n g p o t e n t i a l and i nput r e s i s t a n c e f o r the 65 neurons i nc luded i n t h i s s e r i e s of experiments were -70 (^2.25) mV and 51 (^2.5) M&. These neurons f i r e d s p i ke s of >70 mV ampl itude and were <2 ms i n du r a t i on i n response to d e p o l a r i z i n g cu r ren t pu l s e s . The f i v e - b a r r e l l e d i o n t o p h o r e t i c e l e c t r o d e s which were used here, were e i t h e r glued to the reco rd ing p i p e t t e s or i n s e r t e d sepa ra te l y at the sur face of the s l i c e ( c f . Methods). The reco rd ing m i c r o e l e c t r o d e s were f i l l e d w i t h KCl (3 M) or K 2 S 0 4 (0.6 M). 5.1 R e s u l t s 5.1.1 Con t ro l responses to i o n t o p h o r e t i c a l l y a pp l i ed agents 5.1.1.1 A c e t y l c h o l i n e . A p p l i c a t i o n s of ACh (50-350 nA) evoked a mean (*SEM) d e p o l a r i z a t i o n of 12.3 (-±1.5) mV in 36 out of 50 neurons t h a t were l o ca ted i n an area corresponding to l a y e r s IV and V of the neocortex. However, at more s u p e r f i c i a l l a ye r s (II and I I I ) , ACh a p p l i c a t i o n induced a l o n g - l a s t i n g h y p e r p o l a r i z a t i o n i n 3 neurons ( c f . Wong and Ga l l aghe r 1989). The input r e s i s t a n c e s of the neurons were monitored by r e p e t i t i v e t e s t s us ing cons tant h y p e r p o l a r i z i n g pu l ses and were u s u a l l y i nc reased (~30%) by such a p p l i c a t i o n s ; i n 4 neurons the input r e s i s t a n c e s were decreased, probably due t o membrane (delayed) r e c t i f i c a t i o n . The ACh-induced d e p o l a r i z a t i o n s were slow i n onset as we l l as in decay ( F i g . 17) and were r ep roduc i b l e on repeated a p p l i c a t i o n to the same neuron, w i thout obvious s igns of d e s e n s i t i z a t i o n ( F i g . 17A1). These ac t i on s cou ld be b locked by the a p p l i c a t i o n of scopolamine (20 uM; 3 neurons). Neurons t ha t e x h i b i t e d the l a r g e s t responses to ACh a p p l i c a t i o n u s u a l l y f i r e d sp ikes i n doub let s when i n j e c t e d w i t h d e p o l a r i z i n g cu r r en t pu l se s . These c e l l s had i npu t r e s i s t a n c e H. EL-BEHEIRY 71 A1 A 2 ACh , , —1 I 20 s F i g . 17 E f f e c t s o f i o n t o p h o r e t i c a l l y a p p l i e d a c e t y l c h o l i n e (ACh) on n e o c o r t i c a l neurons. (A) a t y p i c a l l y slow onset response to ACh e j e c ted from a compound e l e c t r o d e assembly i s shown f o r a sensor imotor neuron. (A2) In the same neuron i n j e c t i o n s of j u s t - t h r e s h o l d d e p o l a r i z i n g cu r r en t pu l ses evoked a c t i o n p o t e n t i a l s i n doub le t s . Note the l a r g e amount of spontaneous s ynap t i c a c t i v i t y as we l l as the sagging h y p e r p o l a r i z i n g response to cu r r en t pu l se i n j e c t i o n . (B) response of a sensor imotor neuron to ACh a p p l i e d from a separate e l e c t r o d e c on s i s t ed of a small d e p o l a r i z a t i o n and inc reased s p i k i n g a c t i v i t y which was preceded by a b r i e f h y p e r p o l a r i z a t i o n . (CI) ACh evoked a d e p o l a r i z a t i o n w i t h an a f t e r b u r s t of s ynapt i c a c t i v i t y de sp i te the presence of TTX. H y p e r p o l a r i z i n g vo l t age responses to i n t r a c e l l u l a r c u r r e n t i n j e c t i o n s be fo re , d u r i n g , and a f t e r ACh a p p l i c a t i o n are shown in (C2). H. EL-BEHEIRY 72 of approx imate ly 50 Mn, and e x h i b i t e d a l a r ge amount of spontaneous s ynap t i c a c t i v i t y as we l l as ' sagg ing responses ' t o h y p e r p o l a r i z i n g c u r r e n t pu l se i n j e c t i o n s ( F i g . 17A2). The responses to ACh cou ld be grouped i n t o two main c a t e g o r i e s : (1) d e p o l a r i z i n g response w i t h an a f t e r b u r s t of s ynapt i c a c t i v i t y ( F i g . 17C1) t h a t was observed on severa l occas ions i n the presence o f TTX and (2) d e p o l a r i z i n g response t h a t was preceded by a h y p e r p o l a r i z a t i o n ( F i g . 17B); t h i s h y p e r p o l a r i z i n g p o t e n t i a l which was obta ined when the i o n t o p h o r e t i c e l e c t r o d e was independent from the record ing e l e c t r o d e , cou l d be blocked w i th a p p l i c a t i o n of 1-1.5 nM TTX. 5.1.1.2 Glutamate and N-methy l -D-aspar tate. In c o n t r a s t to the e f f e c t s of ACh, the responses to Glu were abrupt in onset and had a r a p i d o f f s e t , i . e . , were an " on/o f f " type of response (F i g s . 18A-C). A l l 65 neurons were depo l a r i z ed by Glu a p p l i c a t i o n s (40-300 nA) and d i d not e x h i b i t d e s e n s i t i z a t i o n . The mean (±SEM) d e p o l a r i z a t i o n was 20.25 (±1 .4 ) mV and was a s s o c i a t e d w i th a decreased input r e s i s t a n c e of ~40-80% ( F i g . 18A2). Bath a p p l i c a t i o n of TTX had no e f f e c t on the respons iveness of the neurons to Glu ( F i g . 18B; c f . Z ieg lgSnsberger and P u i l 1972). In ~15% of the neurons, a b r i e f pe r i od (2-3 s) of h y p e r p o l a r i z a t i o n (2-4 mV) was observed at the end of the d e p o l a r i z i n g response ( c f . Padjen and Smith 1983). I t was not uncommon f o r the r a t i o of "ACh/Glu d e p o l a r i z a t i o n s " which v a r i ed between 0 .7 -1 , to equal un i t y ( F i g . 18C). There was a tendency towards a h i gher r a t i o when the e l e c t r ode i n the assembl ies had i n t e r t i p d i s t ance s of 50 nm. The Glu-evoked d e p o l a r i z a t i o n s produced by c o n s i s t e n t l y spaced e j e c t i o n pu l se s of equ i v a l en t c u r r e n t were remarkably r ep roduc i b l e de sp i t e the l eng th of the exper imental pe r i od ( e . g . , 3 h r . ) . H. EL-BEHEIRY 73 Glu ACh F i g . 18 Glutamate (Glu) evoked responses i n n e o c o r t i c a l neurons. (A l ) Glu a p p l i c a t i o n induced a f a s t " o n/o f f " type of d e p o l a r i z i n g response which was a s s oc i a t ed w i th an i nc rea sed conductance (A2). (B) the Glu a c t i on s were not a f f e c t e d by the p r e f u s i o n of TTX (1.5 uM). (C) a l t e r n a t i n g a p p l i c a t i o n s of Glu and ACh on an a n t e r i o r c i n g u l a t e neuron produced d e p o l a r i z i n g responses (ACh/Glu r a t i o = ~0.85) which are shown t y p i c a l l y here. H. EL-BEHEIRY 74 S i m i l a r l y b r i e f NMDA a p p l i c a t i o n s depo l a r i z ed 15/37 neurons de sp i t e the +2 presence of 2 mM Mg i n the bath. NMDA induced s l ow l y deve lop ing d e p o l a r i z i n g responses t h a t a l s o had a slow decay ( F i g . 19A); t h i s response was apparent i n c o n j u n c t i o n w i t h an increased i npu t r e s i s t a n c e t ha t waned a f t e r the peak of the p o t e n t i a l response ( F i g . 19A). Two other types of responses a l s o were observed: (1) d e p o l a r i z i n g response t ha t e x h i b i t e d an cute form of d e s e n s i t i z a t i o n dur ing the a p p l i c a t i o n pe r iod ( F i g . 19B; 5 neurons) and (2) a r e l a t i v e l y f a s t G l u - l i k e response t h a t d e s e n s i t i z e d markedly on repeated a p p l i c a t i o n s ( F i g . 19C; 4 neurons). 5.1.1.3 Gamma-aminobutyrate and b a c l o f e n . I ontophoret i c a p p l i c a t i o n of GABA produced two types of responses (36 neurons): (1) a h y p e r p o l a r i z a t i o n which was observed on ly when the compound e l e c t r o d e assembl ies were used (9/9 neurons) and (2) a d e p o l a r i z a t i o n which was evoked when the a p p l i c a t i o n p i p e t t e was separate from the reco rd ing e l e c t r ode (29/29 neurons). The hyperpol a r i z i n g responses (5-9 mV) to GABA were a s s o c i a t e d w i t h decreased input r e s i s t a n c e (40-60%). An acute d e s e n s i t i z a t i o n ( F i g . 20) was observed as we l l dur ing the a p p l i c a t i o n p e r i o d . Moreover, a l o n g - l a s t i n g hyperpo la r -i z a t i o n was ev i den t when the du ra t i on of GABA a p p l i c a t i o n was i nc reased to >10 s ( c f . B l a x t e r e t a l . 1986). The d e p o l a r i z i n g type of response (14-40 mV) was accompanied by an inc rease i n input conductance and cou ld be f u r t h e r subd iv ided i n t o th ree forms: (1) w i thout d e s e n s i t i z a t i o n ( F i g . 21A), (2) w i th acute d e s e n s i t i z a t i o n dur ing the a p p l i c a t i o n pe r i od ( F i g . 21B), and H. EL-BEHEIRY 75 NMDA Glu F i g . 19 NMDA-induced d e p o l a r i z a t i o n s i n sensor imotor l a y e r V neurons. (A) NMDA a p p l i c a t i o n evoked slow d e p o l a r i z i n g response w i t h i n i t i a l i nc rease i n i npu t r e s i s t a n c e ( lower t r a c e s ) . (B) acute d e s e n s i t i z a t i o n e x h i b i t e d by another neuron i n response to NMDA e j e c t i o n from a separate p i p e t t e . (C) a t h i r d type of NMDA a c t i o n was a f a s t G l u - l i k e response. Th is response d e s e n s i t i z e d markedly on repeated a p p l i c a t i o n . GABA NaCl GABA F i g . 20 A h y p e r p o l a r i z i n g response to GABA app l ied from a compound e l ec t rode assembly. (A) acute d e s e n s i t i z a t i o n of the GABA-evoked response i s shown on the l e f t and l o n g - l a s t i n g hyper-p o l a r i z a t i o n on the r i g h t . (B) The hype rpo l a r i z i n g response i n another neuron was blocked by b i c u c u l l i n e a f t e r 5 min and recovery was obta ined a f t e r 20 min at a f l owra te of 2 ml/min. H. EL-BEHEIRY 77 GABA Fig. 21 Different types of depolarizing responses evoked by dendritic application of GABA. Non-desensitizing (A) and acutely desensitizing (B) responses are shown for two sensorimotor neurons. Conductance changes before, at the peak, and after a depolarizing effect of GABA are evident in (C). TTX.(1.5 uM) was applied in (A and B ) . H. EL-BEHEIRY 78 (3) w i th t a chyphy l a x i s on f requent repeated a p p l i c a t i o n s ( F i g . 22C). On severa l occas ions the GABA d e p o l a r i z a t i o n s were a s s o c i a t ed w i t h sp ikes w i th the r i s i n g and/or f a l l i n g po r t i on s of each response ( F i g s . 21C and 22C). In order to determine i f the d e p o l a r i z i n g response was indeed i n h i b i t o r y , i . e . , decreased membrane e x c i t a b i l i t y , Glu was a p p l i e d con t i nuous l y to induce sp ike a c t i v i t y and t h i s was fo l l owed by a concomitant p u l s e - l i k e a p p l i c a t i o n of GABA ( F i g . 22B). The d e p o l a r i z i n g response induced by GABA suppressed the a c t i o n p o t e n t i a l s evoked by Glu a p p l i c a t i o n . In such cases, the i n h i b i t i o n of sp ike f i r i n g was not l i k e l y a t t r i b u t a b l e to excess ive d e p o l a r i z a t i o n because the combined d e p o l a r i z a t i o n was j u s t s up ra th re sho ld . GABA was a p p l i e d to 4 neurons wi th r e s t i n g p o t e n t i a l s t h a t were d i s p l a ced by DC-current i n j e c t i o n to l e v e l s between -100 and -30 mV i n the presence of TTX to determine a r e v e r s a l p o t e n t i a l f o r the GABA d e p o l a r i z a t i o n ( F i g . 22A). The d e p o l a r i z a t i o n s evoked by GABA inc reased i n ampl i tude at more h y p e r p o l a r i z i n g values of the r e s t i n g membrane p o t e n t i a l . However, at an imposed p o t e n t i a l of ~-30 mV the response was m u l t i p h a s i c ( F i g . 22A). A f t e r 4 min of b i c u c u l l i n e (50 uM) a p p l i c a t i o n (3 neurons) the h y p e r p o l a r i z i n g response was blocked ( f l ow r a t e 2 ml/min; F i g . 20 ) . The GABA-evoked d e p o l a r i z a t i o n was b locked only by pro longed a p p l i c a t i o n of b i c u c u l l i n e i n doses as h igh as 150 uM (5 neurons; F i g . 23A). A concomitant e x t r a c e l l u l a r a p p l i c a t i o n of bac lo fen (50 uM) d i d not a l t e r the response to GABA. Bac l o fen when a p p l i e d i o n t o p h o r e t i c a l l y from a separate p i p e t t e d id not change the r e s t i n g p o t e n t i a l or input conductance (6 neurons). However, bath a p p l i c a t i o n o f bac l o f en hype rpo l a r i zed 3 neurons by 2-3 mV w i t h 10-15% inc rease i n conductance and d i d not a f f e c t Glu d e p o l a r i z a t i o n s . I o n t o p h o r e t i c a l l y a p p l i e d bac l o fen d id not depress the number, ampl i tudes or shapes of Glu-evoked sp ikes ( F i g . 23B). H. EL-BEHEIRY 79 15 mV 10 mV GABA GABA 5s 15 mV F i g . 22 C h a r a c t e r i s t i c s neurons. (A) s h i f t i n g D C - i n j e c t i o n revea led a concomitant a p p l i c a t i o n repeated a p p l i c a t i o n of of the GABA-evoked d e p o l a r i z a t i o n s i n n e o c o r t i c a l the r e s t i n g membrane p o t e n t i a l by i n t r a c e l l u l a r b i pha s i c response beg inn ing a t V m <-40 mV. (B) of GABA shunted the sp ikes evoked by G l u . (C) GABA of <10 s i n t e r v a l s d e s e n s i t i z e d the neuron complete ly to the subsequent GABA a p p l i c a t i o n s . The sp ikes GABA responses were probably due to e x c i t a t o r y a c t i o n s of w i t h the a gon i s t . on top of the H + c o - e j e c t e d H. EL-BEHEIRY 80 F i g . 2 3 B i c u c u l l i n e , i n high doses, b locked the GABA- induced d e p o l a r i z a t i o n s i n n e o c o r t i c a l neurons. ( A ) b i c u c u l l i n e (150 M b locked the d e p o l a r i z i n g responses to d e n d r i t i c a p p l i c a t i o n o f GABA whereas the Glu-responses were not a f f e c t e d . (B) b a c l o f e n , a GABAB a g o n i s t , had no e f f e c t on V o r on the Glu-evoked sp ikes i n a sensor imotor neuron. H. EL-BEHEIRY 81 5.1.2 Anae s the t i c - i nduced a l t e r a t i o n s i n responses to e x c i t a t o r y and  i n h i b i t o r y t r a n s m i t t e r substances. The observed changes i n responses t o the e x c i t a t o r y or i n h i b i t o r y t r a n s m i t t e r substances which are de s c r i bed below were not a s soc i a ted w i t h marked changes i n pas s i ve membrane p r o p e r t i e s . For example, on ly the h i ghes t doses of i s o f l u r a n e and A l t h e s i n (2.5 MAC and 200 uM r e s p e c t i v e l y ) h ype rpo l a r i z ed the neurons by 3-5 mV and decreased t h e i r i npu t r e s i s t ance s by <20% . 5.1.2.1 Responses to ACh, Glu and NMDA. I s o f l u r ane and A l t h e s i n a p p l i c a t i o n s (0.5-2.5 MAC; 25-200 uM) depressed the ACh responses i n a dose-dependent and r e v e r s i b l e manner ( F i g s . 24 and 25) . The ECf^s f ° r the e f f e c t s of i s o f l u r a n e and A l t h e s i n were approx imately 0.9 MAC and 75 nM r e s p e c t i v e l y . The suppress ion of the ACh responses was e v i den t a f t e r 6-12 min of anaes the t i c p e r f u s i o n . Depending on the dose, f u l l recovery was observed a f t e r 8-16 min ( f l ow r a t e 1.5-3 ml/min). U s u a l l y , p a r t i a l recovery and in some cases, f u l l recovery was observed on high dose a p p l i c a t i o n of e i t h e r agent (2-2.5 MAC i s o f l u r a n e and 150-200 uM A l t h e s i n ; F i g . 25B). Both anae s the t i c s depressed the G lu - induced d e p o l a r i z a t i o n s i n a dose-dependent and r e v e r s i b l e manner ( F i g . 26 and 27A). The E C 5 Q f o r the i s o f l u r a n e - i n d u c e d a t t enua t i on of the Glu-responses was ~1.9 MAC which i s h i gher than t h a t f o r the corresponding depress ion of ACh-induced d e p o l a r i z a t i o n s . In c o n t r a s t , the A l t h e s i n concen t r a t i on which suppressed the ACh response by 50% depressed the Glu-evoked d e p o l a r i z a t i o n s by about 40%. The G lu- induced responses were p o t e n t i a t e d i n 4 neurons ( F i g . 28) and were not a f f e c t e d i n 10 neurons, by e i t h e r agent. Note t h a t i n F i g . ' 2 8 , i s o f l u r a n e a p p l i c a t i o n enhanced the Glu responses and almost t o t a l l y b locked ACh a c t i o n s . H. EL-BEHEIRY 82 A Control A C h ACh B F i g . 24 Depress ion by i s o f l u r a n e (IFL) of responses to a c e t y l c h o l i n e (ACh; 150 nA f o r 18s i n A , 100 nA f o r 20s i n B) i n 2 neurons (A, V m = -70 mV; B, V m = -68 mV). TTX (1.5 nM) was app l i ed i n A. M idd le records (A,B) were obta ined a t 9 min of IFL a p p l i c a t i o n . H. EL-BEHEIRY 83 15 mV F i g . 25 Depress ion by A l t h e s i n (AL) of responses to ACh (125 nA f o r 9s in A, 95 nA f o r 25s i n B) i n 2 neurons (A, Vm = -72 mV; B, V m = -70 mV). Negat ive vo l t age d e f l e c t i o n s ( t h i c k record i n B) rep re sent t e s t s f o r changes i n i nput r e s i s t a n c e . TTX (1.5 nM) was a p p l i e d i n B. M idd le records were obta ined at 8 min (A) and 6 min (B) of AL a p p l i c a t i o n . H. EL-BEHEIRY 84 20 mV F i g . 26 Depress ion by i s o f l u r a n e ( IFL) of responses to glutamate (G lu ; 100 nA f o r 5s i n A, 95 nA f o r 8s i n B) i n 2 neurons (V m = -70 mV i n A and B) . Negat ive vo l t age d e f l e c t i o n s ( t h i c k record i n B) represent t e s t s f o r changes i n i npu t r e s i s t a n c e . TTX (1 nM) was a p p l i e d i n B. M idd le records were obta ined a t 9 min (A) and 10 min (B) of IFL a p p l i c a t i o n . B Control Glu NMDA AL 1 0 0 ^ M Recovery NMDA IFL 2 MAC F i q 27 Anaes the t i c depress ion of responses to Glu (35 nA) and NMDA (55 nA f o r 5s i n A , 110 nA f o r 15s i n B) i n 2 neurons (V m = -75 mV i n A, -69 mV i n B) . Res i s tance t e s t pu l ses were a p p l i e d on ly i n B. TTX (1.5 pM) was app l i ed i n A. Middle t r ace s were obtained at 10 mm (A) and 9 min (B) of anae s the t i c a p p l i c a t i o n . Voltage c a l i b r a t i o n : 20 mV i n A, 30 mV i n B. H. EL-BEHEIRY 86 F i g . 28 P o t e n t i a t i o n of Glu a c t i on s and a t t enua t i on of ACh responses by i s o f l u r a n e (1MAC) i n an a n t e r i o r c i n g u l a t e neuron. Recovery was ev ident a f t e r 16 min. V m was kept cons tant at -72 mV by cont inuous D C - i n j e c t i o n . H. EL-BEHEIRY 87 Because of the the poor s e n s i t i v i t i e s of the neurons ( l a y e r s IV and V) to i o n t o p h o r e t i c a l l y a pp l i ed NMDA observed i n the presence of e x te rna l +2 Mg (2 mM), a f u l l dose-response r e l a t i o n s h i p cou l d not be con s t ruc ted i n these exper iments . Nonethe les s , both anae s the t i c s a t tenuated the NMDA-evoked d e p o l a r i z a t i o n s i n a dose-dependent manner ( F i g . 27) and t o approx imately the same extent as the G lu - induced responses. A p p l i c a t i o n of halothane (1.5 MAC; 6 neurons) suppressed the G l u - , NMDA- and ACh-induced responses i n a manner s i m i l a r to t h a t observed w i th i s o f l u r a n e a d m i n i s t r a t i o n . 5.1.2.2 Responses to GABA. I s o f l u r ane a d m i n i s t r a t i o n a t 1.5 MAC (5 neurons; F i g . 29A) had no e f f e c t on the h y p e r p o l a r i z i n g responses evoked by GABA a p p l i c a t i o n s . However, 2 MAC i s o f l u r a n e suppressed the responses to GABA by ~15%. A d m i n i s t r a t i o n of i s o f l u r a n e or A l t h e s i n s l i g h t l y reduced the d e p o l a r i z i n g responses t o GABA i n the low dose range (0.5-1.5 MAC i s o f l u r a n e ; 25-100 LIM A l t h e s i n , F i g s . 29B and 30 ) . The maximum depress ion i n these responses was not more t ha t 40% even when doses as h igh as 2.5 MAC i s o f l u r a n e and 200 nM A l t h e s i n were app l i ed ( F i g . 31) . 5.1.3 S e l e c t i v i t y i n the depress ion of responses t o t r a n s m i t t e r  substances 5.1.3.1 Anae s the t i c i n t e r a c t i o n s w i t h t r a n s m i t t e r s . In most neurons anae s the t i c a p p l i c a t i o n produced reduc t i on s i n the responses to both ACh and Glu (Table 1 ) . However, a c e t y l c h o l i n e a c t i o n s were more s u s cep t i b l e to depress ion by e i t h e r i s o f l u r a n e or A l t h e s i n . Th i s s e l e c t i v i t y was most apparent i n neurons where i n i t i a l l y , these t r a n s m i t t e r responses were approx imate ly equal i n amplitude ( F i g s . 29B and 32 ) . The s e l e c t i v i t y was much more pronounced w i t h i s o f l u r a n e than a f t e r A l t h e s i n a p p l i c a t i o n ( F i g . 32 ) and was not observed on pe r fu s i on of the v e h i c l e , cremophor EL. In 7 H. EL-BEHEIRY 88 F i g . 29 E f f e c t s of i s o f l u r a n e ( IFL) a p p l i c a t i o n on equ i - amp l i t ude responses to GABA (100 nA f o r 5s i n A, 75 nA f o r 6s i n B ) , G l u , and ACh i n 2 neurons (V m = -75 mV i n A, -72 mV i n B ) . TTX (1 MM) was a p p l i e d i n A. Res i s tance t e s t pu l se s were a p p l i e d only i n B. Glu (85 nA) and ACh (90 nA) were a p p l i e d i n B. M idd le t r a ce s i n A and B were obta ined at 9 min of IFL a p p l i c a t i o n . Voltage c a l i b r a t i o n : 10 mV i n A, 20 mV i n B. 30 mV 20s m i F i g . 30 E f f e c t s of A l t h e s i n a p p l i c a t i o n on the ACh, GABA and Glu-evoked responses (ACh 70 nA f o r 23s; Glu 90 nA f o r 10s; GABA 110 nA f o r 10s). Note t h a t , i n i t i a l l y , a prominent hyper-p o l a r i z i n g response to ACh was present . Anaesthet ic a p p l i c a t i o n depressed the d e p o l a r i z a t i o n induced by ACh, s l i g h t l y attenuated the Glu-response and had no e f f e c t on the response to GABA. H. EL-BEHEIRY 90 GABA Glu ACh Isoflurane (2 MAC I Z n T l f r T T m ^ ^ t17wrrrTrrrmrHTT 25s 20 mV F i g . 31 Depress ion of ACh, Glu and GABA responses by high dose of i s o f l u r a n e i n neuron (Vm = -68 mV ; ACh 85 nA f o r 10s; Glu 100 nA f o r 5s ; GABA 75 nA f o r 8 s ) . H. EL-BEHEIRY 91 Table 1. Anaes the t i c depress ion of neuronal responses to t r a n s m i t t e r substances Anaes the t i c D e p o l a r i z a t i o n evoked by a p p l i e d  ACh G lu NMDA (n = 36) (n = 65) (n = 15) Hype rpo la r i z a t i o n Depo la r i z a t i o n (n = 9 ) (n = 29) GABA evoked I s o f l u r a n e 18/18 30/40 8/10 4/9 10/17 A l t h e s i n 15/16 25/32 7/9 11/15 Halothane 3/4 5/7 2/4 Depress ion was de f i ned as >15% a t tenua t i on of c o n t r o l responses by the a n a e s t h e t i c . The t o t a l number of the neurons i n v e s t i g a t e d was 65, i . e , a l l neurons were t e s t e d to Glu and to at l e a s t one other t r a n s m i t t e r substance. The anaes the t i c doses were 0.5-2.5 MAC f o r i s o f l u r a n e , 25-200 nM f o r A l t h e s i n and 0.5-2 MAC f o r ha lothane. H. EL-BEHEIRY 92 A, TOO nA i n B ) . Res i s tance t e s t pu l ses were a p p l i e d only i n A. Vo l tage c a l i b r a t i o n : 20 mV i n A, 10 mV i n B. H. EL-BEHEIRY 93 neurons, low dose a d m i n i s t r a t i o n of i s o f l u r a n e o r A l t h e s i n produced no depres s ion of the 61 u - d e p o l a r i z a t i o n and sometimes prolonged the d u r a t i o n of the Glu response ( c f . F i g . 32B). When GABA, Ach and/or G lu were app l i ed a l t e r n a t i v e l y to the same neuron, the a d d i t i o n a l a p p l i c a t i o n of i s o f l u r a n e or A l t h e s i n d i d not s i g n i f i c a n t l y a l t e r the d e p o l a r i z i n g e f f e c t s of GABA, s l i g h t l y reduced those of Glu and markedly attenuated the responses to Ach ( F i g s . 29B-31). 5.1.3.2 Time course of anae s the t i c a c t i o n s . The depress ions produced by i s o f l u r a n e o r A l t h e s i n had r a p i d onset s . The Ach-evoked response u s u a l l y was suppressed a f t e r ~3 min, and was maximal ly reduced a f t e r "8-12 min of anae s the t i c a p p l i c a t i o n ( F i g . 33). In 5 neurons, the reduc t i on s i n the responses to Ach were ev ident a t 30-90s, i . e . , before de te c t ab l e a t tenua t i on of the Glu responses. A d m i n i s t r a t i o n of i s o f l u r a n e , A l t h e s i n or suppressed the responses to NMDA (15 of the 19 neurons) w i th t ime courses t h a t were s i m i l a r to the anaes the t i c r educ t i on s i n Glu responses. F u l l recovery from anae s the t i c e f f e c t s on the Glu d e p o l a r i z a t i o n u s u a l l y was observed severa l minutes e a r l i e r than a complete recovery of the Ach responses to c o n t r o l ampl i tude ( F i g . 33 ) . 5.1.3.3 Dose-response r e l a t i o n s h i p s . The dose-response r e l a t i o n s h i p s f o r the a c t i o n s of i s o f l u r a n e and A l t h e s i n on the jus t -max imal responses evoked by the t r a n s m i t t e r substances are shown i n F i g s . 34 and 35. Each po in t on the curves represent s an averaged depress ion produced by 4 a p p l i c a t i o n s of e i t h e r a n a e s t h e t i c . The EC^ 0 f o r the i s o f l u r a n e depress ion of the Ach responses was 0.9 MAC compared w i t h 1.9 MAC f o r t he suppress ion of Glu responses. Such d i f f e r e n c e s i n s e l e c t i v i t y a l s o were apparent i n a comparison of Glu w i t h the NMDA responses, a l though the data were more l i m i t e d . The s e l e c t i v i t y was l e s s pronounced i n the case H. EL-BEHEIRY 94 100 75--50--25-- Isoflurane (1 MAC) • - • GABA (n=5) A - A Glu (n=6) o-o ACh (n=6) 12 18 TIME (min) F i g . 33 Time courses f o r anae s the t i c depress ion of jus t -max imal responses to GABA, Glu and ACh i n the presence of TTX (1 LIM) . Two or 3 t r a n s m i t t e r substances were a p p l i e d a l t e r n a t i v e l y to each neuron. E r r o r bars i n d i c a t e * S.E. mean. H. EL-BEHEIRY 95 c o o N _o o OL CD Q ~XD CU o > c o CO cu CL CD Q 100 T 80- -60- -40- -20- -O Ach V NMDA 100-r 1-0 2.0 0.5 1.0 2.0 Isoflurane Concentration (MAC) F i g . 34 Pooled data show dose-response r e l a t i o n s h i p s f o r i s o f l u r a n e - i n d u c e d depress ions of d e p o l a r i z a t i o n s evoked by t r a n s m i t t e r substances. Each po in t on the curve i s the mean response to a t l e a s t 4 a p p l i c a t i o n s (n = 22) . Symbols (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t p < 0.05. Ove ra l l s i g n i f i c a n c e was determined by ANOVA. E r r o r bars i n d i c a t e * S .E . mean. H. EL-BEHEIRY 96 F i g . 35 Dose-response r e l a t i o n s h i p s f o r A l t h e s i n - i n d u c e d depress ion o f d e p o l a r i z a t i o n s evoked by t r a n s m i t t e r substances. Each po i n t on the curve i s the mean response to at l e a s t 4 a p p l i c a t i o n s (n = 20) . Symbols (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t p < 0.05. O v e r a l l s i g n i f i c a n c e was determined by ANOVA. E r r o r bars i n d i c a t e * S .E . mean. H. EL-BEHEIRY 97 o f A l t h e s i n where the E C 5 0 s were 75 nM f o r the depress ion of the ACh responses and 90 uM f o r the depress ion of Glu responses ( F i g . 35). The maximal reduct ions in the d e p o l a r i z i n g responses to GABA a p p l i c a t i o n were on ly about 40% at 2.5 MAC i s o f l u r a n e o r 200 uM A l t h e s i n . The e f f e c t s of anae s the t i c s were f u r t h e r q u a n t i f i e d i n cases where the ~ED 5 Q o f a t r a n s m i t t e r substance had been app l i ed to the same neuron ( F i g . 36A). the a ce t y l cho l i ne/g l u t amate response r a t i o (ACh/Glu) was depressed more by i s o f l u r a n e than by A l t h e s i n a p p l i c a t i o n , a lthough the ACh-evoked d e p o l a r i z a t i o n s were more s u s c e p t i b l e to the depressant a c t i o n s of both a n a e s t h e t i c s . This s u s c e p t i b i l i t y a l s o was revea led by i n v e s t i g a t i o n s of anae s the t i c e f f e c t s on the ju s t -max ima l responses to ACh, Glu and GABA which were a p p l i e d f o r comparison i n the same neurons ( F i g . 36B). 5.1.3.4 Anae s the t i c e f f e c t s on the p o t e n t i a t i o n of G lu- responses  by ACh. Th i s p o s s i b i l i t y was examined i n 7 neurons by app ly ing Glu ( " E D J J Q ) f o r 6s dur ing longer a p p l i c a t i o n s of ACh ( ~ E D 5 Q ) l a s t i n g 120-180 s, and then concomitant ly app l y i ng the anaes the t i c ( i s o f l u r a n e i n 4, and A l t h e s i n i n 3 neurons) . As can be seen from F i g s . 37 and 38, the a p p l i c a t i o n of ACh enhanced the e f f e c t s of Glu i . e . , the d e p o l a r i z a t i o n s were a d d i t i v e but there was a q u a l i t a t i v e change i n the d i s charge which appeared t o be d i s p r o p o r t i o n a t e t o the magnitude of the combined d e p o l a r i z a t i o n . When 1.5 MAC i s o f l u r a n e or 75 nM A l t h e s i n was a d d i t i o n a l l y adm in i s t e red , t h i s enhancement was no longer e v i den t . Note t h a t such anae s the t i c a p p l i c a t i o n s a l s o reduced the G lu responses which recovered a t about the same time as the ACh e f f e c t s from the anae s the t i c dep re s s i on . 5.2 D i s cu s s i on 5.2.1 Responses t o t r a n s m i t t e r and r e l a t e d agents. A l though there were no fundamental d i f f e r e n c e s between the respons iveness of n e o c o r t i c a l neurons H. EL-BEHEIRY 98 Isoflurane (1.5 MAC) Althesin (75 /xM) F i g . 36 S e l e c t i v i t y i n i s o f l u r a n e and A l t he s i n -evoked depress ion of neuronal respons iveness to ACh, Glu and GABA. (A) ACh/Glu r a t i o of d e p o l a r i z i n g responses was depressed by anaes thet i c a p p l i c a t i o n s i n d i c a t i n g g r e a t e r s u s c e p t i b i l i t y of the ACh responses. E r r o r bars show ± S . E . mean. Symbols (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l at p < 0.05. (B) pooled data from 12 neurons i n which at l e a s t 2 t r a n s m i t t e r substances evoked jus t -max imal response. E r r o r bars show ±S .E . mean. Post -hoc comparison t e s t was used. Symbols (*) i n d i c a t e s i g n i f i c a n t d i f f e r e n c e from c o n t r o l a t p < 0.05 as determined by S t uden t ' s t - t e s t . Ove r a l l s i g n i f i c a n c e was determined by ANOVA. H. EL-BEHEIRY 99 IFL 11.5 MACI ACh 25 mV ACh F i g . 37 Continuous reco rd of i s o f l u r a n e ( I FL ) - i nduced depres s ion of the A C h - p o t e n t i a t i o n of G l u - a c t i o n s . D e p o l a r i z a t i o n s evoked by Glu ( c f . negat ive a r t e f a c t s at onset of 70 nA cu r ren t ) and ACh (50 nA) are ~ 50% of t h e i r maximal responses i n t h i s neuron. IFL was a p p l i e d f o r 5 min ( t h i c k b a r ) . H. EL-BEHEIRY 100 Glu ACh Althesin 1 0 0 MM ACh 30mV ACh 60s F i g . 38 A l t h e s i n - i n d u c e d depress ion o f the ACh -po ten t i a t i on of G l u - a c t i o n s . D e p o l a r i z a t i o n s evoked by Glu (60 nA) and ACh (40 nA) were depressed by A l t h e s i n a p p l i c a t i o n at 8 min; recovery was ev i den t a f t e r 15 min ( lower t r a c e ) . H. EL-BEHEIRY 101 i n these i n v e s t i g a t i o n s and the s e n s i t i v i t i e s of other neuronal c e l l types i n o ther areas of the CNS ( K r n j e v i c 1974b) to the t r a n s m i t t e r substances, some q u a l i t a t i v e d i f f e r e n c e s have been i d e n t i f i e d by these i n v e s t i g a t i o n s . 5.2.1.1 A c e t y l c h o l i n e . The d e p o l a r i z a t i o n and inc reased i npu t r e s i s t a n c e induced by ACh were de sc r i bed p r e v i o u s l y f o r n e o c o r t i c a l neurons i n decerebrate ( K r n j e v i c e t a l . 1971b) and awake ca t s (Woody et a l . 1978). In n e o c o r t i c a l s l i c e p r e p a r a t i o n s , McCormick and P r ince (1986) a l s o observed t h a t ACh evoked a r a t he r small l o n g - l a s t i n g d e p o l a r i z a t i o n w i t h sp i ke bu r s t f i r i n g t h a t was preceded by a b r i e f h y p e r p o l a r i z a t i o n and an i nc reased conductance. The i n i t i a l h y p e r p o l a r i z a t i o n was a t t r i b u t e d to a c t i v a t i o n of an i n te rneuron by the ACh " p u f f e d " from an independent m i c r o p i p e t t e because i t c ou l d be blocked s e l e c t i v e l y by concomitant TTX a p p l i c a t i o n . In the p resent i n v e s t i g a t i o n s , both types of responses were observed a f t e r ACh a p p l i c a t i o n from two types of e l e c t r o d e placements. The p o s s i b i l i t y of a c t i v a t i n g an interneuron i s l i k e l y to be l e s s w i th the compound e l e c t r o d e assembl ies having i n t e r t i p d i s t ance s of ~50 um because the t r a n s m i t t e r i s e j e c t e d most ly near the soma of the neuron ( P u i l 1974). With such assembl ies on l y s low d e p o l a r i z a t i o n s were observed, whereas ACh a p p l i c a t i o n from an independent i o n t o p h o r e t i c p i p e t t e , evoked a s h o r t - l a s t i n g h y p e r p o l a r i z a t i o n which cou l d be blocked w i t h TTX and was always f o l l owed by a d e p o l a r i z i n g response. The " a f t e r b u r s t " of s ynapt i c a c t i v i t y recorded on ly o c c a s i o n a l l y i n the presence of TTX ( c f . F i g . 17C) cou ld be due to p re synapt i c a c t i o n s on n i c o t i n i c receptor s of nerve t e rm ina l s (Ropert and K r n j e v i c 1982; Rov i r a e t a l . 1983) or spontaneous r e l ea se of e x c i t a t o r y t r a n s m i t t e r s . Not a l l the neurons (presumably pyramidal c e l l s ) , were respons ive t o ACh a p p l i c a t i o n s (~70% were respons ive) de sp i t e t h e i r h igh s e n s i t i v i t i e s t o G l u . Pa r t of t h i s A C h - i n s e n s i t i v i t y cou ld be exp la i ned by the damage of the H. EL-BEHEIRY 102 neurons produced by e l e c t r o d e pene t r a t i on by the r e co rd i n g e l e c t r o d e , p a r t i c u l a r l y i n view of the a c t i o n of ACh to inc rease neuronal i npu t r e s i s t a n c e by dec reas ing membrane conductance f o r K + . F u n c t i o n a l d i f f e r e n c e s between neurons recorded i n v i t r o and those recorded i n the i n t a c t or decerebrate p repa ra t i on s where the pathways are und i s tu rbed may prov ide o ther reasons f o r the i n s e n s i t i v i t y observed i n 30% of the neurons. The r e l a t i v e l y l o n g - l a s t i n g e f f e c t s of ACh app l i ed on the neurons ( c f . Re su l t s ) may be a t t r i b u t a b l e t o a long-term modulat ion o f musca r in i c secondary messenger systems ( c f . Woody and Gruen 1988; M u l l e r e t a l . 1988; Dutar and N i c o l l 1988). 5.2.1.2 Glutamate and N-methy l -D-aspar tate. An out s tand ing f ea tu re i n the G lu - induced responses was t h e i r con s i s t ency both q u a l i t a t i v e l y and q u a n t i t a t i v e l y de sp i t e the l ength of the exper imenta l pe r i od ( e . g . , 3-4 hour s ) . U n l i k e G lu , NMDA-evoked d e p o l a r i z a t i o n s e x h i b i t e d both acute and long-term forms of d e s e n s i t i z a t i o n , probably due to the +2 presence of ex te rna l Mg (Mayer and Westbrook 1987; MacDonald e t a l . 1987). The occas iona l d i f f i c u l t y in induc ing equ i v a l en t responses to Glu and ACh ( e s p e c i a l l y i n the case of a p p l i c a t i o n s from the compound e l e c t r o d e assembl ies ) cou ld be exp l a i ned by the known topograph ica l d i s t r i b u t i o n of the g lu tamaterg i c and c h o l i n e r g i c ( i n t h i s case, musca r i n i c ) r e cep to r s . For example, the l o c a t i o n s of high s e n s i t i v i t y or the " ho t spots " f o r Glu may be on dend r i t e s whereas those f o r ACh are l o ca ted main ly on the soma. , 5.2.1.3 Y-aminobuty r a t e . GABA a p p l i c a t i o n s to n e o c o r t i c a l neurons produced hyperpol a r i z i n g and d e p o l a r i z i n g responses. The hyperpol a r i z i n g responses cou ld be evoked on ly by GABA a p p l i c a t i o n from the compound e l e c t r o d e s and were r e a d i l y b locked by b i c u c u l l i n e (50 uM); these cou ld H. EL-BEHEIRY 103 r e s u l t from i n t e r a c t i o n s w i th somatic GABAA r ecep to r s ( c f . Bormann 1988). The d e p o l a r i z i n g responses observed e x c l u s i v e l y w i th the separate a p p l i c a t i o n and reco rd ing e l e c t r o d e s are probably a t t r i b u t a b l e to s t i m u l a t i o n of another subpopulat ion of r ecep to r s f o r GABA t h a t are l o c a t e d on the dend r i t e s of the pyramidal neurons ( c f . A l g e r and N i co l 1982). D e p o l a r i z i n g responses to GABA have been repor ted i n the sp ina l cord (Barker and Ransom 1978b; C u r t i s and Gynther 1987) and a f t e r d e n d r i t i c a p p l i c a t i o n i n the hippocampus (Andersen e t a l . 1980; Thalmann e t a l . 1981; A l g e r and N i co l1 1982; A v o l i and P e r r e a u l t 1987; B l a x t e r and Ca r len 1988). In c o r t i c a l neurons t h i s type of response can be b locked by b i c u c u l l i n e and i s mediated most ly by an i n c rea se i n C I " conductance ( K rn j e v i c and Schwartz 1967; A l g e r and N i co l 1 1979, 1982; Andersen e t a l . 1980; Thalmann et a l . 1981; M i s ge ld et a l . 1986b). In the present i n v e s t i g a t i o n s , the GABA d e p o l a r i z a t i o n s were only b locked by l a r g e doses (>100 LIM) of b i c u c u l l i n e and then on ly a f t e r prolonged a p p l i c a t i o n s (>15 min ; 2ml/min f low r a t e ) . Moreover, a r e v e r s a l p o t e n t i a l of these responses cou ld not be a t t a i ned when the r e s t i n g p o t e n t i a l was s h i f t e d by DC-current i n j e c t i o n i n the range of -100 t o -30 mV i n the presence of TTX ( c f . F i g . 21C). Desp i te the u n c e r t a i n i t i e s as to the i o n i c mechanisms and the subtype of GABA-receptor mediat ing t h i s response, the response evoked by GABA was capable of " s h u n t i n g " sp ikes e l i c i t e d by a p p l i c a t i o n of Glu to the same neuron. In severa l neurons sp i ke a c t i v i t i e s were observed on the r i s i n g and f a l l i n g phases of the d e p o l a r i z i n g response to GABA; s i m i l a r a c t i o n s of GABA may, u n d e r l i e a pa radox i ca l e x c i t a t o r y phenomena de sc r i bed e a r l i e r by P u i l e t a l . (1974) i n the i s o l a t e d n e o c o r t i c a l s l ab i n v e s t i g a t i o n s or repor ted f o l l o w i n g s t i m u l a t i o n of c o r t i c a l neurons by r e l ea se of H + c oncomi tan t l y w i t h the GABA e j e c t i o n s ( c f . K r n j e v i c and Schwartz 1967). H. EL-BEHEIRY 104 5.2.2 Anae s the t i c - i nduced a l t e r a t i o n s i n the c h e m o s e n s i t i v i t y of  e x t r a c e l l u l a r l y a p p l i e d t r a n s m i t t e r agents. A l though K r n j e v i c and Phi 11 i s (1963a) found many yea r s ago, t h a t ce reb ra l c o r t i c a l neurons showed a lower s e n s i t i v i t y t o ACh i n b a r b i t u r a t e - a n a e s t h e t i z e d animals i n comparison to the cerveau i s o l i p r e p a r a t i o n s , only a few i n v e s t i g a t i o n s r epo r t ed l y have exp lo red the anae s the t i c - i nduced a l t e r a t i o n s i n the respons iveness of n e o c o r t i c a l neurons t o va r ious t r a n s m i t t e r s and r e l a t e d substances. Subsequent r epo r t s by K r n j e v i c and h i s group repor ted t ha t a wide v a r i e t y of int ravenous and v o l a t i l e anaes the t i c s s e l e c t i v e l y depressed the e x c i t a t o r y a c t i o n s of ACh wh i l e the G l u - and the GABA-responses were preserved i n decerebrate animals ( K r n j e v i c and Phi 11 i s 1963b, c ; K r n j e v i c e t a l . 1966; Catch love e t a l . 1972; K r n j e v i 6 and P u i l 1975; P u i l and K r n j e v i c 1978). However, t h i s p r e f e r e n t i a l a t tenuat i on of ACh a c t i on s on n e o c o r t i c a l c e l l s has not been conf i rmed by other i n v e s t i g a t o r s (Crawford and C u r t i s 1966; Crawford 1970; c f . B a z i l and Minnemann 1989). Moreover, halothane o r t h i o p e n t a l a p p l i c a t i o n have been shown to depress, u n i f o rm l y , the e x c i t a t o r y responses e l i c i t e d by a p p l i c a t i o n s of ACh or monoamines (5 -hydroxyt ryptamine, no radrena l i ne and i s o p r e n a l i n e ) i n n e o c o r t i c a l neurons of decerebrate ca t s (Johnson e t a l . 1969). A major d i sadvantage i n the use of e x t r a c e l l u l a r reco rd ing techniques f o r u n i t d i s cha rge a c t i v i t y i n the abovementioned r epo r t s i s the inherent problem w i th the i n t e r a c t i o n s of e f f e c t s from nearby neurons e x c i t e d by the t r a n s m i t t e r a p p l i c a t i o n s . Th i s may p rov ide a reason f o r the d i s s i m i l a r i t i e s w i t h the r e s u l t s of the present s t u d i e s . Another reason may be r e l a t e d to the va ry ing degrees t h a t the i n t a c t ascending i n h i b i t o r y and e x c i t a t o r y pathways r a d i a t e to the ce reb ra l c o r t e x as w e l l as t o the v a r i a b i l i t y i n the e f f e c t s o f d i f f e r e n t anae s the t i c s on the c e r e b r a l blood f l o w of the i n t a c t an ima l s . H. EL-BEHEIRY 105 U n f o r t u n a t e l y , on ly incomplete i n f o rmat i on i s a v a i l a b l e about the e f f e c t s of anae s the t i c s on the chemical respons iveness of o ther neurons a t d i f f e r e n t l e v e l s i n the c e n t r a l neurax i s of mammals. In the c e r e b e l l a r c o r t e x of i n v i vo c a t p r epa r a t i o n s , b a r b i t u r a t e s s e l e c t i v e l y depress ACh-evoked e x c i t a t i o n without a t tenua t i ng Glu a c t i on s on spontaneously d i s cha r g i n g neuronal u n i t s recorded e x t r a c e l l u l a r l y (McCance and P h i l l i s 1964). Desp i te the suppress ion of the e f f e c t s of ACh, noradrena l ine and s e r o t o n i n on u n i t responses of the o l f a c t o r y bulb i n i n v i vo exper iments , i n h a l a t i o n a l agents depress G l u - f i r i n g and p o t e n t i a t e A C h - e x c i t a t i o n recorded e x t r a c e l l u l a r l y i n the o l f a c t o r y co r tex i n v i t r o (R ichards and Smaje 1976; Smaje 1976). Responses to G l u , Quis , ka and NMDA in hippocampal CA1 neurons i n v i t r o are suppressed to va ry ing degrees by b a r b i t u r a t e s (Sawada and Yamamoto 1985). However, G l u - and G A B A - e l i c i t e d d e p o l a r i z a t i o n s are not a f f e c t e d by halothane or i s o f l u r a n e a p p l i c a t i o n i n low doses. Higher doses of these agents depressed on ly the Glu-responses and d i d not have any e f f e c t on the responses t o GABA (Brooks e t a l . 1986). B a r b i t u r a t e s and va r i ou s e ther s a t tenuate ACh- and not G lu - induced d i scharges i n the thalamus ( P h i l l i s and Tebec i s 1967) and basal g ang l i a (Bloom e t a l . 1965; Spencer and Hav l i c ek 1974). Bra instem neurons and sp i na l motoneurons e x h i b i t a s e l e c t i v e decrease i n t h e i r c h e m o s e n s i t i v i t i e s to ACh app l i ed i n t e r m i t t e n t l y w i t h monoamines and Glu (Bradley and Dray 1973). In terpeduncu lar neuronal responses to ACh are l e s s a f f e c t e d than substance P-evoked e x c i t a t i o n s dur ing anae s the t i c a p p l i c a t i o n of in t ravenous or v o l a t i l e agents ( Sas t ry 1978). The A C h - f a c i l i t a t i o n of u n i t a c t i v i t y of medul lary neurons (Sa lmoi ragh i and S t e i n e r 1963) and Renshaw c e l l s (B i scoe and K r n j e v i c 1963) i s not a t tenuated by anae s the t i c s admin i s tered i n i n v i vo c a t p repa ra t i on s . A l though the H. EL-BEHEIRY 106 a v a i l a b l e evidence may be i n t e r p r e t e d on ly w i th some u n c e r t a i n t i e s , the p o s s i b i l i t y t h a t anae s the t i c s have s e l e c t i v e e f f e c t s on the s e n s i t i v i t i e s to suspected t r a n s m i t t e r s on s u b c o r t i c a l neurons t h a t have some re levance to the p roduct ion of the anae s the t i c s t a t e should be subjected to e x ten s i v e r e - i n v e s t i g a t i o n . In the present exper iments , the use of the i n v i t r o s l i c e p repa ra t i on and TTX-blockade of Na-sp ike genes is f o r the i n v e s t i g a t i o n s of anae s the t i c m o d i f i c a t i o n s i n the chemical respons iveness of the n e o c o r t i c a l neurons reduced po s s i b l e i n h i b i t o r y or e x c i t a t o r y i n f l uence s ex te rna l to the recorded neuron. D i f f e r ence s i n neuronal metabol ism i n v i t r o and i n v i vo a l s o should be cons idered in comparing these r e s u l t s w i th the i n fo rmat i on obta ined from i n t a c t animals. An i n t e r e s t i n g ob se r va t i on i n the present s t u d i e s i s the g r e a t e r a t tenua t i on by i s o f l u r a n e or A l t h e s i n a p p l i c a t i o n , of the ACh-induced responses compared to the G l u - or the NMDA-evoked d e p o l a r i z a t i o n s . Under c o n d i t i o n s of s ynapt i c blockade by TTX and in the absence of s i g n i f i c a n t changes induced by the anae s the t i c i n the pas s i ve membrane p r o p e r t i e s , the suppress ion of the c h e m o s e n s i t i v i t y of the neurons to the e x c i t a t o r y t r a n s m i t t e r agents c ou l d be due t o anaes thet i c e f f e c t s on the i o n i c channe l - r e cep to r complex or on the modulat ion of the secondary messenger system re spons i b l e f o r the a gon i s t / r ecep to r b ind ing impulse t r a n s d u c t i o n . Recent pa t ch - and vo l tage-c lamp s tud ie s on d i f f e r e n t systems ( L e c h l e i t e r and Gruener 1984; L e c h l e i t e r e t a l . 1986; Ar imura and Ikemoto 1986; F l an i g an and B r e t t 1987; Ikemoto e t a l . 1988) suggest t h a t the c h o l i n e r g i c depress ion produced by anae s the t i c s may r e s u l t from a decrease i n the maximum number of channel s a c t i v a t a b l e by ACh. This may be due to a s t a b i l i z a t i o n of the c l o s e d s t a t e of the channel or to an a l l o s t e r i c H. EL-BEHEIRY 107 m o d i f i c a t i o n of the channel p r o t e i n s lead ing to a sho r ten ing of the r a te cons tant s governing t r a n s i t i o n s between the open s t a t e and one or more normal ly o c c u l t c l o sed s t a t e s , w i th a subsequent excess i ve f l i c k e r i n g of the ionophore. B ind ing s t u d i e s have shown that a wide range o f genera l anaes-t h e t i c s b ind w i th the ACh-receptor a l l o s t e r i c a l l y and i n h i b i t the s p e c i f i c b ind ing or s t a b i l i z e the h i g h - a f f i n i t y s t a t e of the r e c e p t o r ; the outcome of these a c t i o n s probably corresponds to the non -de sen s i t i z ed s t a t e in the i n v i vo s i t u a t i o n (Young and Sigman 1981). In a d d i t i o n , halothane has been found to conver t G p r o t e i n - c o u p l e d and -uncoupled musca r i n i c r ecep to r to s t a t e s of lower agon i s t a f f i n i t y and to lower the a f f i n i t y of receptor -G p r o t e i n complexes f o r guanine nuc leo t i de s (Aronstam e t a l . 1986; Dennison e t a l . 1987; Anthony e t a l . 1989). On the other hand, the depress ion of the G lu - i nduced d e p o l a r i z a t i o n s by i s o f l u r a n e and A l t h e s i n cou ld be a t t r i b u t e d in pa r t to an i nc reased a f f i n i t y of Glu to i t s r e cep to r s and p a r t l y to a decrease i n the maximal number of a c t i v a t a b l e recepto r -channe l complexes which would r e s u l t in the format ion of e i t h e r a b l o c ked - c l o s ed or d e s e n s i t i z e d s t a t e ( c f . Ikemoto e t a l . 1988). However, i n the absence of suppor t i ve b iochemical and patch-c lamp data such suggest ions remain h i g h l y s p e c u l a t i v e . More r e c e n t l y , P u i l and Baimbridge (1989) observed t h a t i s o f l u r a n e attenuated the Glu-evoked i nc rease in [Ca ].. i n c u l t u r e d hippocampal neurons. They concluded t h a t the a c t i o n s of i s o f l u r a n e and halothane most l i k e l y i n vo l ve a b lockade of Ca-channels a c t i v a t e d by i n t e r a c t i o n s of Glu wi th e i t h e r Quis o r NMDA types of recepto r s and a d i r e c t b lockade of vo l tage -ga ted Ca-channels. A suppress ion of the GABA-evoked d e p o l a r i z a t i o n s has been repor ted f o r p e n t o b a r b i t a l i n doses h igher than 300 nM, anaes thet i c doses of a lphaxa lone, and high doses of en f l u r ane (Connors 1980; Simmonds 1981; C u l l e n and H. EL-BEHEIRY 108 M a r t i n 1982; Parker e t a l . 1986; Brooks e t a l . 1986). The reasons f o r the e f f e c t s observed p r e v i o u s l y and i n the present s t u d i e s are not c l e a r . P o s s i b i l i t i e s i n c l ude the p lugg ing of the c h l o r i d e channel s by high concen-t r a t i o n s of the a n a e s t h e t i c , s i m i l a r to t h a t observed a t ACh -ac t i va ted endplate channe l s (Adams 1976) or the occupat ion of the a c t i v e s i t e s on the GABA recep to r by the anaes thet i c molecu les . Q u a l i t a t i v e l y , the present i n v e s t i g a t i o n s are concordant w i th the r e s u l t s of e x t r a c e l l u l a r s t ud i e s performed many yea r s ago on mammalian n e o c o r t i c a l neurons i n v i vo ( K r n j e v i c and P h i l l i s 1963a,b,c; Catch love e t a l . , 1972). The observed dose-response r e l a t i o n s h i p s a l l u d e t o the p o s s i b i l i t y t ha t the anae s the t i c depress ions of e x c i t a t o r y and i n h i b i t o r y t r a n s m i t t e r ac t i on s in the i n t a c t animal are a continuum of i n t e r f e r e n c e beginning w i t h ACh-suppress ion and ending wi th complete a t tenua t i on of the Glu (and NMDA-) responses. Th i s i s i n c on t r a s t to the gene ra l i z ed concept of depress ion of the c h e m o s e n s i t i v i t i e s of CNS neurons dur ing anae s the s i a , and suggests much more s e l e c t i v e anae s the t i c a c t i o n s . The r e s u l t s of these experiments are not i n agreement w i t h the observa-t i o n s i n o l f a c t o r y c o r t i c a l neurons where i n h a l a t i o n a l anae s t he t i c s d i d not depress u n i t d i s cha rge evoked by ACh a p p l i c a t i o n (Smaje 1976). In a d d i t i o n to the i nhe rent d i f f e r e n c e s in the membrane e x c i t a b i l i t i e s of n e o c o r t i c a l and o l f a c t o r y neurons, the low doses of anae s the t i c s a p p l i e d t o the o l f a c t o r y c o r t i c a l s l i c e s may e x p l a i n the d i s c repan t r e s u l t s . In the o l f a c t o r y i n v e s t i g a t i o n s , ha lothane, d i e t h y l e ther and t r i c h l o r o e t h y l e n e had been admin i s te red i n doses of <0.5 MAC values f o r rodent s . Increas ing the concen t r a t i on s of these agents i n the atmosphere of the s l i c e suppressed the f i r i n g r a t e of the neurons ( c f . F i g . 4 in Smaje 1976). H. EL-BEHEIRY 109 In c o n c l u s i o n , the c h e m o s e n s i t i v i t i e s of n e o c o r t i c a l neurons i n the i n v i t r o p repa ra t i on are reduced by a p p l i c a t i o n s of i s o f l u r a n e or A l t h e s i n i n a s e l e c t i v e and gradual manner; these e f f e c t s begin wi th ACh and end w i t h complete a t t enua t i on of the ACh- and Glu- responses approx imately a t the anae s the t i c ED^Q and EDg^s r e s p e c t i v e l y . Th is s e l e c t i v e a t t enua t i on o f the ACh-induced responses a t low doses, w i t h p r e s e r v a t i o n of the G l u - d e p o l a r i z a t i o n s , may r e f l e c t d i f f e r e n t a f f i n i t i e s f o r the anae s the t i c molecu les to t h e i r s i t e s (membrane recep to r s ) of a c t i o n . The mechanism of such an i n t e r f e r e n c e may be r e l a t e d to the channe l - r ecep to r complex p r o t e i n and/or to the secondary messenger systems re spon s i b l e f o r the a gon i s t -r e cep to r b ind ing impulse t r a n s d u c t i o n . 6 EFFECTS OF HYPOMAGNESIA ON TRANSMITTER AND ANAESTHETIC ACTIONS 6.1 Re su l t s 2+ The e f f e c t s of a r educ t i on i n the [Mg ] were i n v e s t i g a t e d i n 28 sensor imotor neurons on t h e i r responses to e x t r a c e l l u l a r a p p l i c a t i o n s o f ACh, G lu and GABA be fo re , du r i n g , and a f t e r an a p p l i c a t i o n of i s o f l u r a n e or A l t h e s i n . These neurons had a mean V m and R^  r e s i s t a n c e of -76.3 (±5.97; ±S.D.) mV and 44.96 (±16.46) Mn, r e s p e c t i v e l y . I n t r a c e l l u l a r c u r r e n t i n j e c t i o n s evoked sp ikes w i t h ampl i tudes of more than 80 mV and du ra t i on s of l e s s than 2 ms. The c u r r e n t - v o l t a g e curves i n 85% of the neurons revea led inward r e c t i f i c a t i o n . A p p l i c a t i o n s of ACh, Glu and GABA (50-300 nA f o r 4-20 s) produced mean d e p o l a r i z a t i o n s of 13.12 (± 3 .6 ) , 22.5 (± 8.7) and 20.5 (± 6.8) mV r e s p e c t i v e l y . The i nput r e s i s t a n c e s of these neurons were e i t h e r inc reased (~15%) o r una f f ec ted near the peak H. EL-BEHEIRY 110 d e p o l a r i z i n g response induced by ACh. Input r e s i s t a n c e s were always decreased (35-90%) by Glu or GABA a p p l i c a t i o n s de sp i t e the presence (n = 18) or absence (n = 10) of TTX (1-1.5 uM). 6.1.1 E f f e c t s of bath a p p l i c a t i o n of Mg-free ACSF. The c a l c u l a t e d 2+ [Mg ] i n the nomina l l y Mg-free media was ~ l - 3 uM because of t r a c e i m p u r i t i e s i n the s a l t s used (Mayer and Westbrook 1985; Sutor e t a l . 1987). Pe r f u s i on of the s l i c e s w i t h t h i s "Mg- f ree " s o l u t i o n , i n the presence or absence of TTX, induced s l i g h t h y p e r p o l a r i z a t i o n (3-5 mV) i n 8 neurons. However, the R- o f the neurons were e i t h e r unchanged or decreased by ~10% (5/8 and 3/8 neurons r e s p e c t i v e l y ) . In the absence o f the Na + -channe l b l ocke r the spontaneous and the background synapt i c a c t i v i t i e s were g r e a t l y enhanced. A f t e r ~ l - 3 hrs i n cuba t i on i n nomina l ly Mg-free medium and TTX pe r f u s i o n abrupt pe r i ods of spontaneous slow d e p o l a r i z i n g waves were recorded i n 4 neurons ( c f . F i g . 39A). A p p l i c a t i o n s of ACh (5/6 neurons) and GABA (10/10 neurons) induced d e p o l a r i z a t i o n s t h a t were reduced i n Mg-free s o l u t i o n s w i th or w i thout TTX present ( F i g . 39B). On the o the r hand, Glu responses were i n c o n s i s t e n t l y a f f e c t e d dur ing pe r f u s i on w i th s o l u t i o n s c on t a i n i n g zero mM Mg. The d e p o l a r i z a t i o n s evoked by Glu were e i t h e r depressed ( F i g s . 39B and 40) unchanged o r , on other occa s i on s , p o t e n t i a t e d . Responses to NMDA t h a t were comparable to the G lu responses cou l d not be obta ined dur ing pe r f u s i o n w i t h c o n t r o l s o l u t i o n (10/10 neurons). Dur ing cont inuous pe r f u s i on of Mg-free medium, a b r i e f a p p l i c a t i o n of NMDA evoked a d e p o l a r i z a t i o n of ~20 mV i n 6/10 neurons ( F i g . 40 ) . These r e s u l t s are summarized i n Table 2. 2+ 6.1.2 E f f e c t s of gradual removal of [Mg ] . The e f f e c t s o f d i f f e r e n t l e v e l s of hypomagnesia, were s tud ied i n s l i c e s t h a t were g r a d u a l l y 2+ dep le ted from t h e i r i n i t i a l e x t r a c e l l u l a r Mg content by sequent i a l H. EL-BEHEIRY 111 0 mM Mg 1 5 m V ACh Glu GABA F i g . 39 Blockade of ACh and GABA ac t i on s i n Mg-free p e r f u s a t e . (A) spontaneous d e p o l a r i z i n g waves evoked i n a sensor imotor neuron (V m = -70 mV) from a n e o c o r t i c a l s l i c e incubated i n M g 2 + - f r e e media. (B) ACh and GABA responses were at tenuated i n neuron (V m = -75 mV) by removal of M g 2 + from the media. V e r t i c a l bar i n d i c a t e s 15 mV i n B. H. EL-BEHEIRY 112 Glu NMDA I OmM Mg+ 2 1 JL M TTX A A A 2 0 m V 4 0 s F i g . 40 P o t e n t i a t i o n of NMDA responses i n the Mg-free bath ing s o l u t i o n c on t a i n i n g TTX. NMDA-responses were g r e a t l y p o t e n t i a t e d , wh i l e the Glu-evoked d e p o l a r i z a t i o n s were depressed. H. EL-BEHEIRY 113 Table 2 E f f e c t s of Mg-free media on sensor imotor neurons* ** Depress ion of *** P o t e n t i a t i o n of Glutamate Responses ACh responses GABA responses NMDA responses Depres- No change Po t en -s ion t i a t i o n 7/7 13/13 10/10 3/9 4/9 2/9 * H y p e r p o l a r i z a t i o n was observed i n 8/16 of the neurons. A decrease i n i n pu t r e s i s t a n c e ( ± 1 0 % ) was detected i n 5/16 neruons. 7\ /VDepress ion was de f i ned as >20% a t t enua t i on of c o n t r o l ampl i tudes of ACh-or GABA-induced d e p o l a r i z a t i o n . P o t e n t i a t i o n was de f ined as >20% increase of c o n t r o l ampl i tudes o f g lutamate or NMDA. H. EL-BEHEIRY 114 2+ pe r f u s i on of ACSF s o l u t i o n s c o n t a i n i n g low [Mg ] . The e f f e c t s of removal of the Mg-cat ion from the e x t r a c e l l u l a r environment on the Glu induced d e p o l a r i z a t i o n s are shown in F i g . 41. A gradual depress ion of the Glu responses were not observed i n other neurons (7/10) t ha t showed no change or a s l i g h t p o t e n t i a t i o n of the Glu a c t i o n s . However, the e f f e c t s of GABA were c o n s i s t e n t l y reduced by the gradual 2+ d e p l e t i o n of Mg i n a dose-dependent manner i n 11/11 neurons. Recovery was slow but u s ua l l y was observed w i t h i n the exper imental p e r i o d . A s i n g l e 2+ a p p l i c a t i o n of low Mg s o l u t i o n (0.5 mM) f o r 15-20 min produced an a t t e n -ua t i on of the GABA responses t h a t was complete ly r e v e r s i b l e ( F i g . 42A). On 2+ the o the r hand, i n c r e a s i n g the [Mg ] to 3.5 mM i n the pe r fu s i ng media had no e f f e c t on the GABA-evoked d e p o l a r i z a t i o n s ( F i g . 42B). 2+ 6.1.3 E f f e c t s of i n c r e a s i n g [Mg ] . Neurons (n = 8) in s l i c e s t h a t were incubated i n Mg-free ACSF s o l u t i o n f o r 3-7 hours showed ex ten s i ve spontaneous and s ynapt i c a c t i v i t i e s . During pe r f u s i on w i t h i n c r e a s i n g 2+ [Mg ] the neurons e x h i b i t e d a corresponding p rog re s s i ve suppress ion o f the exaggerated spontaneous f i r i n g and synapt i c background which had been present j u s t a f t e r impalement. A s l i g h t d e p o l a r i z a t i o n (±4 mV) which was not a s s o c i a t ed w i t h a change i n the i nput r e s i s t a n c e was observed i n 6/8 neurons. Linder these c o n d i t i o n s the neurons d i d not respond to ACh a p p l i c a -2+ t i o n s . However, sequent ia l pe r f u s i on w i th 0.5, 1 and 2 mM Mg concen t r a -t i o n s produced a steady i nc rease i n the ACh-evoked d e p o l a r i z a t i o n s (5/8 neurons; F i g . 43 ) . A p p l i c a t i o n s of Glu evoked d e p o l a r i z i n g responses i n a l l neurons (8/8). However, the Glu responses were una f fec ted (4/8) or 2+ enhanced (4/8) by the sy s temat ic i nc rease i n [Mg ] ( c f . F i g . 43 ) . The GABA-induced d e p o l a r i z a t i o n s were g r a d u a l l y p o t e n t i a t e d in a dose-dependent 2+ manner by the increments i n [Mg ] ( F i g . 44 ) . 2mM Mg 1 mM Mg 0.5 mM Mg 0 mM Mg 15min 2mM Mg 20 min 20 mV 45 s , j 9 _ *Jr«™\.™?*\<*. ^ o . i n a neuron i\i « r r w. «,u . c ^u. . i C i ujr y rduud i removal or L^ g Jo m a neurc o m M M 2+7 C o m P l e t e r e c°very was obtained after 20 min in control solutions (c mM Mgc ) -m r— i CD 3 H. EL-BEHEIRY 116 A F i g . 42 Depress ion of GABA-evoked d e p o l a r i z a t i o n by low [ M g 2 + ] 0 . (A) pe r f u s i o n w i th 0.5 mM M g 2 + c on ta i n i n g media depressed the responses to GABA i n an a n t e r i o r c i n g u l a t e neuron (V m = -68 mV). (B) i n c r e a s i n g l e v e l s of [ M g 2 + ] 0 had no e f f e c t on GABA-induced d e p o l a r i z a t i o n s . H. EL-BEHEIRY 117 Mg-free Glu ACh • • 20 s F i g . 43 Gradual p o t e n t i a t i o n of ACh-act ions i n a neuron (V m = -70 mV) due to a sequent i a l i n c rea se of [ M g 2 + ] 0 . Recordings were obta ined i n a s l i c e t h a t had been incubated i n f r e e Mg-media f o r ~3 hours. V e r t i c a l bar i n d i c a t e s 15 mV. H. EL-BEHEIRY 118 2 5 m V 30 s F i g . 44 GABA a c t i o n s were g r a d u a l l y p o t e n t i a t e d by sequent ia l i nc rea se i n [Mg* J 0 c oncen t r a t i on i n the pe r f u s i n g media. Recordings were obta ined from a neuron i n a s l i c e tha t had been incubated i n Mg-free ACSF f o r ~4 hours. H. EL-BEHEIRY 119 The magnitudes of the d e p o l a r i z a t i o n s evoked by Ach, Glu and GABA at 2+ each [Mg ] were averaged i n 18 neurons ( F i g . 4 5 ) , The changes i n the responses to ACh and GABA were l a r g e r and approached t h e i r r e s p e c t i v e maxima 2+ sooner w i th i n c r e a s i n g l e v e l s of [Mg ] . However, the r a t e of r i s e and 2+ magnitude of the Glu responses were l a r g e l y independent of the [Mg ] . 2+ 2+ 6.1.4 E f f e c t s of e x te rna l Ca and Mg e x c l u s i o n on the Glu 2+ responses. Because of the v a r i a b i l i t y in the e f f e c t s of Mg removal and bu i ldup on the Glu responses, a separate set of exper iments was designed to exp lo re the a l t e r a t i o n s i n Glu-responses by man ipu la t i ng the d i v a l e n t c a t i o n environment w i thout changing the t o t a l c a t i o n i c c o n c e n t r a t i o n . These were 2+ 2+ c a r r i e d out i n the presence of TTX. E xc l u s i on of both Ca and Mg 2+ from the pe r fu s i ng media and t h e i r s u b s t i t u t i o n s w i th 4 mM Co at tenuated the Glu- induced d e p o l a r i z a t i o n s by 38.8 (±7.5)% i n 3 out of 4 neurons 2+ 2 + ( F i g . 46A). S u b s t i t u t i o n of Ca w i th Co suppressed the Glu responses by 15 (±5 .7 )% i n 4 out of 4 neurons ( F i g . 46B). Recovery was complete a f t e r 15-20 min. 6.1.5 Anaes the t i c a c t i o n s dur ing Mg-free p e r f u s i o n . Anae s t he t i c s were a p p l i e d a f t e r pe r f u s i n g the s l i c e w i th Mg-free s o l u t i o n f o r 25-35 min. I s o f l u r ane (1.5 MAC; 4 neurons) a p p l i c a t i o n s suppressed the G l u - and GABA-evoked d e p o l a r i z a t i o n s by 35.5 (±10)% and 55.4 ( ±7 . 2 )% r e s p e c t i v e l y ( F i g . 47A). A l t h e s i n (75 nM; 5 neurons) at tenuated G l u - and GABA-induced responses by 32.8 (±6 .4 )% and 51.2 ( ±8 .5 )% r e s p e c t i v e l y ( F i g . 47B). The GABAergic a c t i o n s were more vu lne rab le to suppress ion by the anae s the t i c s than the responses evoked by G l u . Th is was i n c o n t r a s t to the obse rva t ions 2+ obta ined i n 2 mM Mg ( c f . Sec t ions 5.1 and F i g s . 34 and 35 ) . However, i n neurons where ACh e l i c i t e d d e p o l a r i z a t i o n s of 5-8 mV ampl i tude i n zero mM 2+ Mg , i s o f l u r a n e complete ly b locked the e f f e c t s of ACh ( c f . F i g . 47A). H. EL-BEHEIRY 120 30 - i > OH , , , 0 1 2 3 M g + 2 c o n c e n t r a t i o n ( m M ) F i g . 45 Dose-response curves showing the e f f e c t s of [ M g 2 + ] 0 on t he d e p o l a r i z a t i o n s evoked by ACh, Glu and GABA. Raw data were obta ined from 18 neurons subjected to d i f f e r e n t l e v e l s of [ M g 2 + ] 0 . Control B Ca&Mg-free 4mM Co + 2 Recovery Ca-free 2mM Mg"*? 2mMCo + 2 2 0 m V 3 0 s F i g . 46 E f f e c t s of changing Ca- and Mg-concentrat ions on glutamate ac t i on s i n two neurons (V m = -68 mV) i n A and -79 mV i n B. Responses Glu were depressed i n Ca- and Mg-free s o l u t i o n s (A) more than i n the absence of C a 2 + alone (B) . ro m 3 H. EL-BEHEIRY 122 GABA Glu F i g . 47 Anaes thet i c a c t i o n s on ACh-, G l u - and GABA-induced responses dur ing a p p l i c a t i o n of Mg-free ACSF i n two neurons (V m = -65 mV i n A and -70 i n B ) . I s o f l u r ane ( IFL) and A l t h e s i n (AL) depressed GABA- more than G lu - re sponses . The r e s i d u a l A C h - d e p o l a r i z a t i o n i n (A) was complete ly b locked by A l t h e s i n . C a l i b r a t i o n bars i n d i c a t e 25 s i n A and 60 s i n B, and 20 mV i n A and B. H. EL-BEHEIRY 123 6.2 D i scus s ion A l though man i f e s t a t i on s of h y p e r e x c i t a b i l i t y of the CNS t h a t are apparent c l i n i c a l l y dur ing hypomagnesaemia mod i f i e s the anae s t he t i c requirements p e r i o p e r a t i v e l y (Mar t i nda le and Heaton 1964; Agus et a l 1982; A l d r e t e 1987; Gambling e t a l . 1988), the unde r l y i ng pa thophy s i o l o g i c a l mechanisms have not y e t r ece i ved much a t t e n t i o n . In these exper iments, hypomagnesia was produced 2+ 2+ by s u b s t i t u t i n g Ca f o r Mg i n the pe r fu s ing media such t h a t the t o t a l d i v a l e n t c a t i o n c oncen t r a t i o n was kept constant (4 mM). Consequent ly, any i n t e r f e r e n c e w i t h the su r f ace charge screening of the membrane t h a t can f a c i l i t a t e the a c t i v a t i o n of inward cu r ren t s was avoided (Frankenhauser and Hodgkin 1957; L l i n a s and Walton 1980; Mayer et a l . 1984). As a r e s u l t , a l t e r a t i o n s in the pa s s i ve and a c t i v e membrane p r o p e r t i e s induced by the s imulated hypomagnesaemia are almost e n t i r e l y due to changes i n the 2+ CM9 ] Q (Frankenhauser and Hodgkin 1957; McLaughl in e t a l . 1971; Luttgau and G l i t s c h 1976). U n l i k e hippocampal neurons (Mody et a l . 1987), exposure of the n e o c o r t i c a l s l i c e s to Mg-free media hyperpo la r i zed the neurons i n these i n v e s t i g a t i o n s and decreased t h e i r input r e s i s t a n c e s . In c o n t r a s t , d e p o l a r i z a t i o n s of comparable magnitude were observed on severa l occas ions 2+ dur ing the Mg - bu i l dup exper iments. The l a t t e r e f f e c t s were probably due 2+ to the i n t e r f e r e n c e w i th a t o n i c b l ock ing a c t i on of Mg on C a - a c t i v a t e d K-channels ( L l i n a s and Walton 1980; c f . I s e r i and French 1984 and Begen i s i ch 1988). Accord ing to t h i s scheme, the Ca-channels would be r e l i e v e d from the susta ined blockade by Mg-cat ions r e s u l t i n g i n an enhancement of the r e s t i n g Ca - a c t i v a ted K-conductance and thereby a h y p e r p o l a r i z a t i o n . 6.2.1 Suppress ion of the ACh-induced responses. The dose-dependent 2+ suppress ion of the ACh-induced responses by removal of Mg from the media H. EL-BEHEIRY 124 has not been repor ted p r e v i o u s l y f o r n e o c o r t i c a l neurons. However, i o n t o -2+ pho re t i c a p p l i c a t i o n s of Mg have been found t o depress the ACh- and carbacho i -evoked responses recorded e x t r a c e l l u l a r l y i n the sp i na l cord and dor sa l root gang l i a (Davies and Watkins 1977; Evans and Watkins 1978). 2+ Recent r epo r t s de sc r i be a blockade of ACh a c t i on by a r educ t i on i n [Mg ] 2+ i n c a rd i a c muscle (Kurach i et a l . 1986; 1988) and a f a i l u r e of Mg to antagonize n i c o t i n i c a c t i o n s on c e r e b e l l a r neurons (Garza e t a l . 1987). In neocortex, the depres s ion of ACh responses i n the present i n v e s t i g a t i o n s cou ld be a t t r i b u t e d to one or a combinat ion of the f o l l o w i n g mechanisms: 2+ (1) a decrease i n [Mg ] would remove the t o n i c i n h i b i t i o n of an inward ly d i r e c t e d Ca conductance, thereby i n c r e a s i n g Ca-dependent K - e f f l u x (Wanke et a l . 1987); t h i s would decrease the ACh-induced depres s ion of the i npu t conductance and the t ime- and voltage-dependent M-cur rent , 2+ (2) decreas ing [Mg ] may cause a decrement i n i n t r aneu rona l 2 + [Mg ] (Baker and Crawford 1972; Heinonen and Akerman 1986) thereby enhancing the inward ly r e c t i f y i n g K-conductance which would i n t e r f e r e w i t h the a c t i o n of ACh ( c f . K rn jev i c ' et a l 1967 and S t a n f i e l d 1988), 2+ (3) low [Mg ] f avour s the convers ion of the muscar in i c receptor w i t h h igh a f f i n i t y b ind ing s i t e s to low a f f i n i t y confo rmat iona l s t a t e s ; t h i s i s suggested by the observed s h i f t to the l e f t of the occupancy-c o n c e n t r a t i o n curves f o r carbachol b ind ing i n the c e r e b r a l co r tex 2+ whenever Mg was added t o the i ncubat ing medium (Hulme et a l . 1980; Gurwi tz and Sokolovsky 1980; B i r d s a l l e t a l . 1984; Aronstam e t a l 1985). 2+ 6.2.2 Changes i n [Mg ] do not s i g n i f i c a n t l y a f f e c t G lu- induced  responses. P rev ious i n t r a c e l l u l a r i n v e s t i g a t i o n s i n r a t n e o c o r t i c a l s l i c e s showed no s u b s t a n t i a l changes i n the responses evoked by Glu dur ing H. EL-BEHEIRY 125 a p p l i c a t i o n of Mg-free s o l u t i o n s (Thomson e t a l . 1985; Sutor e t a l . 1987). 2+ In the f r o g and r a t sp ina l co rds , a l t e r a t i o n s of [Mg ] Q i n both d i r e c -t i o n s had no app rec i ab l e e f f e c t s on the i n t r a - o r e x t r a c e l l u l a r l y recorded Glu responses (Evans e t a l . 1977; A u l t e t a l . 1980; Lacey and N i s t r i 1988). The pooled data i n the present s tud ie s i n d i c a t e d a t rend towards s l i g h t depres s ion of the d e p o l a r i z a t i o n s evoked by G l u ; t h i s was not , however, s t a t i s t i c a l l y s i g n i f i c a n t . The absence of an e f f e c t cou ld be due to an 2+ i n a b i l i t y of Mg t o s i g n i f i c a n t l y i n f l u e n c e the b ind ing of Glu to i t s r ecepto r s (Baudry and Lynch 1979) and/or may r e f l e c t Glu i n t e r a c t i o n s predominant ly w i t h Quis and ka (not NMDA s i t e s ) s e n s i t i v e s i t e s at l a y e r s IV-V of the co r tex as suggested by severa l au to rad iog raph ic s t ud i e s (Greenamyre et a l . 1985; Monaghan and Cotman 1985). The observed t rend i n depres s ion of the Gl u-responses, i f conf i rmed by sy s temat ic s t u d i e s , would 2+ suggest t h a t Mg cou ld be a charge c a r r i e r c o n t r i b u t i n g to the Glu-evoked 2+ d e p o l a r i z a t i o n . The decrease i n e x t r a c e l l u l a r Mg a c t i v i t y recorded w i t h i o n - s e l e c t i v e e l e c t r o d e s dur ing l o c a l a p p l i c a t i o n of Glu to hippocampal pyramidal neurons supports t h i s p o s s i b i l i t y (Pumain e t a l . 1987; 1988). In a d d i t i o n , the l a r g e r depress ion of the G lu responses observed i n the absence 2 2+ 2+ of Mg and Ca than i n the presence of Mg i n the pe r fu s i ng media 2+ ( c f . Re su l t s ) a l s o may be i n t e r p r e t e d t h a t Mg c a t i o n s , when p re sen t , permeated through G lu -operated ionophores. 6.2.3 GABA-depo la r i za t i ons are c o n s i s t e n t l y a t tenuated by the removal 2 + 2 + of [Mg 2Q> Em9 ] has been suggested t o be an e s s e n t i a l f a c t o r f o r the p reven t i on of "rundown" i n GABA^-receptor f u n c t i o n ( S t e l z e r e t a l . 1988; Gyenes e t a l . 1988). The e f f e c t s of [Mg 2 + ] on p o s t s y n a p t i c a l l y GABA-induced C l ~ - c u r r e n t s have not been p r e v i o u s l y repor ted f o r mammalian 2+ c e n t r a l neurons. However, a wide v a r i e t y of d i v a l e n t c a t i o n s (Zn , H. EL-BEHEIRY 126 2+ 2 + 2 + Co , Ni and Cd ) antagonize responses t o GABA i n d i f f e r e n t v e r t e -b ra te spec ies (Kaneko and Tachibana 1986). In the absence of c o n c l u s i v e 2+ b iochemica l evidence t ha t [Mg ] a f f e c t s the b ind ing c h a r a c t e r i s t i c s of GABA A r e cep to r s , i t may be assumed t h a t the dose-dependent i n h i b i t i o n of the d e p o l a r i z a t i o n s produced by GABA a p p l i c a t i o n s i n the present i n v e s t i g a -2+ t i o n s i s due t o a decrease i n the i n t r a c e l l u l a r Mg c oncen t r a t i o n 2+ 2 + ([Mg 3^) subsequent to the removal of [Mg ] (Baker and Crawford 1972; Heinonen and Akerman 1986; c f . Fry 1986) which leads to a "rundown" o f GABA^ recepto r f u n c t i o n . 2+ 6.2.4 Enhancement of neuronal e x c i t a b i l i t y by the removal of [Mg ] . 2+ The r educ t i on of [Mg ] Q i s known t o enhance neuronal e x c i t a t i o n i n many c o r t i c a l systems i n c l u d i n g hippocampus and e n t o r h i n a l co r tex (Walter et a l . 1986; Stanton e t a l . 1987; Mody et a l . 1987; Hamon e t a l . 1987; Tancredi e t a l 1990) as we l l as neocortex (Thomson 1986; A v o l i et a l . 1987; Aram et a l . 1989). Severa l mechanisms have been proposed f o r these a c t i o n s , i n c l u -d i n g : 2+ (1) removal of a n t a g o n i s t i c a c t i on s of Mg on p re - and po s t s ynap t i c Ca ent ry (Katz and M i l e d i 1969; L l i n a s and Walton 1980; Lambert and Heineman 1986; Czeh and Somjen 1989), (2) reduced su r face charge screen ing and an a s soc i a ted f a c i l i t a t i o n of inward cu r ren t s and a c t i on p o t e n t i a l s (Frankenhaeuser and Hodgkin 1957; L l i n a s and Walton 1980; Mayer e t a l . 1984) and (3) removal of the voltage-dependent Mg-blockade of NMDA r ecep to r a s s o c i a t ed ionophores (Davies and Watkins 1977; Thomson 1986; Wal ter e t a l . 1986; Mody e t a l . 1987; Stanton e t a l . 1987; MacDonald e t a l . 1987; Mayer et a l . 1988;). H. EL-BEHEIRY 127 In the present i n v e s t i g a t i o n s , the p o t e n t i a t i o n of the NMDA responses i n 2+ low [Mg ] i s c o n s i s t e n t w i t h the above c o n s i d e r a t i o n s . An a d d i t i o n a l mechanism tha t may c o n t r i b u t e t o the neuronal h y p e r e x c i t a b i l i t y under c o n d i t i o n s of hypomagnesia i s the dose-dependent depress ion of the GABA-ergic t r an sm i s s i o n ; thus , the pyramidal neurons would be r e l i e v e d from the negat ive feedback loop i n h i b i t i o n by i n te rneu rons . 6.2.5 Anae s the t i c - i nduced depress ion of Glu and GABA responses i n 2 + Mg - f r e e media. These i n v e s t i g a t i o n s demonstrate, f o r the f i r s t t ime, the depressant a c t i o n s of i s o f l u r a n e and A l t h e s i n on the G l u - and GABA-evoked d e p o l a r i z a t i o n s i n s imulated hypomagnesaemia. An i n t e r e s t i n g f i n d i n g was the reversed p a t t e r n i n suppress ion of the t r a n s m i t t e r - i n d u c e d responses. Both anaes the t i c agents p r e f e r e n t i a l l y depressed GABA- compared w i t h G l u - a c t i o n s . Whereas i n normal [Mg ] , the amount of a t t e n u a t i o n of the GABA-responses was i n s i g n i f i c a n t a t s i m i l a r anae s the t i c c oncen t r a t i o n s . Though the mechanism of such " s e l e c t i v i t y " i s not c l e a r , these observat ions p a r t l y e x p l a i n the increased anae s the t i c requirements i n p a t i e n t s s u f f e r i n g from c o n d i t i o n s a s soc i a ted w i th hypomagnesaemia, e . g . , i n c h r o n i c a l c oho l i sm . 7 ANAESTHETIC-INDUCED ATTENUATION OF POSTPIKE AFTERHYPERPOLARIZATIONS  MEDIATED BY Ca-ACTIVATED K-CONDUCTANCE 7.1 Resu l t s The 52 neurons s e l e c t e d f o r anaes thet i c a p p l i c a t i o n s i n t h i s s e r i e s of experiments had r e s t i n g p o t e n t i a l s more negat ive than -65 mV, sp ike ampl i tudes u s u a l l y of 70-75 mV, and a b i l i t y to f i r e 5 such sp i ke s r e p e t i -H. EL-BEHEIRY 1 2 8 t i v e l y dur ing c u r r e n t - p u l s e i n j e c t i o n s . The depths of s ucce s s fu l impalements corresponded to l a y e r s IV and V of t y p i c a l c o r t e x . The i npu t r e s i s t a n c e s (mean ± SD; 6 5 . 7 ± 2 0 . 5 Mn) and r e s t i n g p o t e n t i a l s ( - 7 3 . 2 ± 7 . 9 mV) remained s t a b l e f o r per iods of 5 0 t o 2 2 0 min. 7 . 1 . 1 Depress ion of AHPs. The e f f e c t s of anae s the t i c a p p l i c a t i o n on the AHPs accompanying 5 ( i n a few cases , 6 ) sp ikes evoked by c u r r e n t - p u l s e i n j e c t i o n s ( c f . Methods) were s tud ied i n the 5 2 neurons. The c o n t r o l AHPs ranged from 3 . 5 - 1 1 mV i n ampl itude and 7 0 0 - 2 8 0 0 ms i n d u r a t i o n . A p p l i c a t i o n s of i s o f l u r a n e or A l t h e s i n produced dose-dependent, r e v e r s -i b l e depress ions i n the ampl itudes and dura t ions of the AHPs in a l l cases ( F i g s . 4 8 - 5 0 ) . These e f f e c t s were observed dur ing DC-compensation f o r any anae s the t i c evoked changes ( u s u a l l y 2 - 5 mV, i f present) i n r e s t i n g p o t e n t i a l ( c f . Methods). I s o f l u r a n e admin i s tered at 1 MAC reduced the mean ampl i tude by ~ 2 3 * 4 . 8 1 % and the mean du ra t i on by ~ 2 6 ± 4 % ( F i g . 5 0 ) . A t an i s o f l u r a n e concen t r a t i on of 2 . 5 MAC, the mean ampl itude and du ra t i on of the AHPs were reduced by ~ 6 8 % and ~ 7 5 % , r e s p e c t i v e l y ( F i g . 5 0 ) . Complete recovery was observed 9 - 1 5 min a f t e r d i s c o n t i n u i n g i s o f l u r a n e a p p l i c a t i o n . The E C ^ Q f o r a t t enua t i on of the AHP ampl itude by A l t h e s i n a p p l i c a t i o n was 1 5 0 nM. The ECgQ f o r the shor ten ing of AHP du ra t i on s was 7 0 L I M which corresponds approx imately to the concen t r a t i on s determined i n r a t b r a i n a f t e r a s i n g l e int ravenous dose of A l t h e s i n t h a t induces an anae s the t i c s t a te i n v i v o (Smith e t a l . , 1 9 7 4 ) . Recovery was observed at 1 0 t o 1 2 min a f t e r te rm ina t i ng a p p l i c a t i o n s of 1 5 - 3 0 0 nM doses. Although A l t h e s i n a p p l i c a t i o n s at the 1 5 0 0 nM dose reduced the mean ampl i tude and du r a t i on by 8 5 . 5 * 1 3 . 6 % and 9 5 . 5 ± 1 8 . 5 % , r e s p e c t i v e l y ( F i g . 5 0 ) , no recovery was observed even at 3 0 min a f t e r r e tu rn i ng to c o n t r o l pe r f u s i on at a r a t e of 3 ml/min ( F i g . 4 9 , l a s t p a n e l ) . The percent changes i n the ampl itude and du ra t i on of AHPs were s i g n i f i c a n t s t a t i s t i c a l l y a t a l l doses of both agents ( F i g . 5 0 ) . H. EL-BEHEIRY 129 F i g . 48 I s o f l u r ane induced an a t tenua t i on of the a f t e r h y p e r p o l a r i z a t i o n s (AHPs). I s o f l u r ane a p p l i c a t i o n s to three neurons i n 0 .5, 1.5, 2.5 MAC doses produced dose-dependent and r e v e r s i b l e reduct ions i n the du ra t i on and ampl-i t ude of the AHPs. controls Isoflurane 0.5MAC recoveries controls Althesin recoveries 15x10" 6 M F ig . 49 Depression of the afterhyperpolarizations (AHPs) by appl ications of A l thes in . The effects were evident at d i f fe rent doses (within two log units of alphaxalone content). The depression by the highest dose of Althes in was i r rever s ib le even after 30 min in control so lut ion. F i g . 50 Dose-response r e l a t i o n s h i p s f o r the depress ion of the a f t e r h y p e r p o l a r i z a t i o n s produced by a p p l i c a t i o n s of i s o f l u r a n e (6-9 min; A,C) and A l t h e s i n (4-6 min; B,D). The data po i n t s were f i t t e d by 3rd order reg re s s i on a n a l y s i s . Symbols ( • ) i n d i c a t e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s from the mean con t r o l values at p < 0.05. H. EL-BEHEIRY 133 7.1.2 B i c u c u l l i n e - b l o c k a d e of i n h i b i t o r y po s t s ynap t i c p o t e n t i a l s B i c u c u l l i n e a p p l i c a t i o n s were used i n 8 non-spontaneously a c t i v e neurons to reduce " c on t am ina t i on " of the AHPs by s ynap t i c and DC p o t e n t i a l s generated through C l -conductances mediated by recepto r s f o r endogenous GABA ( c f . McCormick and P r i n ce 1986). For example, a summation of AHPs w i th IPSPs cou ld have occur red i f the c u r r e n t - p u l s e i n j e c t i o n s had a c t i v a t e d , through r e cu r r en t c o l l a t e r a l s , a s y n a p t i c a l l y connected i n te rneuron t h a t r e l ea sed GABA onto the recorded neuron. A 50 uM c o n c e n t r a t i o n of b i c u c u l l i n e was chosen because of observat ions t h a t IPSPs evoked by e p i c o r t i c a l e l e c t r i c a l s t i m u l a t i o n were complete ly b locked i n such s l i c e p repa ra t i on s ( c f . S e c t i o n 2 ) . The doses of i s o f l u r a n e (1.5 MAC) and A l t h e s i n (100 uM) were chosen f o r f a s t recovery t imes as we l l as f o r t h e i r c l i n i c a l re levance (Smith et a l . 1974; Cu l l en 1986). B i c u c u l l i n e a p p l i c a t i o n alone d i d not g r e a t l y a f f e c t V m and f a c i l i t a t e d r e p e t i t i v e sp i ke burs t s evoked on c u r r e n t - p u l s e i n j e c t i o n s . A small i nc rea se {~10%) i n Rj was observed dur ing b i c u c u l l i n e a p p l i c a t i o n . When i s o f l u r a n e or A l t h e s i n was app l i ed concomi tant l y w i t h b i c u c u l l i n e , the AHPs were reduced much more than wi th the so le a d m i n i s t r a t i o n of the anae s the t i c ( F i g . 51 ) . The anaes thet i c a c t i o n s on the AHPs had approx imate ly the same t ime courses i n the absence and presence o f b i c u c u l l i n e ( F i g . 52) . The changes i n V m and R. induced by A l t h e s i n were l e s s i n t he presence of b i c u c u l l i n e than i n i t s absence ( F i g . 52B). 7.2 D i s cu s s i on The most s t r i k i n g e f f e c t s of both agents on a l l neurons i n t h i s s e r i e s of i n v e s t i g a t i o n s were the dose-dependent a t tenua t i on s i n the ampl i tudes , as we l l as the s h o r t e r du ra t i on s of the AHPs a s s oc i a t ed w i t h the r e p e t i t i v e sp ike d i s cha r ge s . These po s t s ynapt i c e f f e c t s cou ld not be a t t r i b u t e d to H. EL-BEHEIRY 134 F i g . 51 I s o f l u r ane ( IFL ; 1.5 MAC f o r 6 min) and A l t h e s i n (AL; 100 nM f o r 4 min) induced depress ions of the a f t e r h y p e r p o l a r i z a t i o n s (AHPs) i n two neurons (A,B) where b i c u c u l l i n e methiodide (BIC; 50 nM) had been a d d i t i o n a l l y a p p l i e d . The AHPs were a t tenuated more i n the presence of b i c u c u l l i n e than i n i t s absence. Note t h a t a depress ion of the AHPs occur red w i t h the a p p l i c a t i o n of b i c u c u l l i n e a lone. F i g . 52 The mean changes i n r e s t i n g membrane p o t e n t i a l , input r e s i s t a n c e , and a f t e r h y p e r -p o l a r i z a t i o n (AHP) amplitude or du ra t i on in e i gh t neurons were induced by i s o f l u r a n e (A; I FL , 1.5 MAC f o r 6-9 min, n = 4) and A l t h e s i n (B; AL, 100 uM f o r 4-6 min, n = 4 ) . Note the pronounced e f f e c t s of IFL on the AHP in the presence of b i c u c u l l i n e (BIC) i n A. (B) b i c u c u l l i n e p a r t i a l l y blocked the changes i n r e s t i n g p o t e n t i a l and input r e s i s t a n c e , and p o t e n t i a t e d the depress ion of the AHP induced by A l t h e s i n . H. EL-BEHEIRY 136 anae s the t i c induced changes i n r e s t i n g membrane p r o p e r t i e s and a l s o have been observed dur ing s i m i l a r a p p l i c a t i o n s of i s o f l u r a n e to hippocampal CAj c e l l s ( Fu j iwara et a l 1988; Miu and P u i l 1989) and human sympathetic gang l i on neurons ( P u i l e t a l . 1988). A depress ion of d i r e c t l y or s y n a p t i c a l l y - e v o k e d AHPs has been demonstrated du r ing a p p l i c a t i o n s of other v o l a t i l e anaesthe-t i c s , and b a r b i t u r a t e s (Bosnjak e t a l . 1982; Werz and Macdonald 1985; Fu j iwa ra et a l . 1988; Miu and P u i l 1989; Southan and Wann 1989). In some of the present s t ud i e s , b i c u c u l l i n e was admin i s tered concomi tan t l y w i t h the anae s the t i c s to block the GABA-mediated CI-conductances ( c f . McCormick and P r i n c e 1986). Hence, the l i k e l y summations of IPSPs w i t h the AHPs as we l l as w i th endogenous GABA-actions t h a t may have been p o t e n t i a t e d by the a n a e s t h e t i c , e s p e c i a l l y by A l t h e s i n (Barker et a l . 1987) cou ld be prevented i n n e o c o r t i c a l neurons. B i c u c u l l i n e a p p l i c a t i o n alone decreased the ampl i tudes and du ra t i on s of the AHPs suggest ing a blockade of the endogenous GABAergic a c t i v i t y . In such cases , A l t h e s i n a p p l i c a t i o n produced a g r ea te r b lockade of AHPs, presumably because an o c c l u s i o n of the AHP-conductances due t o anaes thet i c p o t e n t i a t i o n of GABA-ergic a c t i v i t y had been avo ided. Both anaes the t i c s had much more pronounced e f f e c t s on the AHPs under c o n d i t i o n s of blockade by b i c u c u l l i n e , suggest ing some o c c l u s i o n of the C a - a c t i v a t e d K-conductance ( c f . Schwindt e t a l . 1988a, 1988b) by a GABA-mediated CI-conductance i n neurons where the GABA^-antagonist had not been a p p l i e d . I t i s u n l i k e l y t h a t a non-Ca-dependent K-conductance i n t e r f e r e d w i t h , or c o n t r i b u t e d t o , the genes i s of the AHPs ( c f . Schwindt e t a l . 1988a, 1988b). Therefore , the e f f e c t s observed i n these i n v e s t i g a t i o n s cou ld be a t t r i b u t e d mainly to anae s the t i c a c t i o n s d i r e c t l y on the i o n i c mechanisms generat ing the AHPs. H. EL-BEHEIRY 137 7.2.1 Mechanism(s) of anaes the t i c i n t e r f e r e n c e w i th AHP gene ra t i on . Normal l y , the AHP i s a consequence of a K-conductance t ha t i s i n i t i a t e d by d e p o l a r i z a t i o n and Ca -en t r y . As a r e s u l t , [Ca ] . r i s e s , a c t i v a t e s c e r t a i n K-channels ( c f . K r n j e v i c ' et a l . 1978b) and i n i t i a t e s an outward K - cu r ren t . The decay of t h i s cu r r en t i s p a r t l y con t i ngen t on the bu i ldup of 2+ [Ca ] . t h a t i n a c t i v a t e s the Ca-channels (Eckert and Chad 1984) and on i n t e r n a l C a - s e q u e s t r a t i o n . Halothane, A l t h e s i n and b a r b i t u r a t e s dep le te i n t r a c e l l u l a r energy s t o re s thereby compromising C a - b u f f e r i n g mechanisms (Smith e t a l . 1974; M o r r i s 1986; Daniel1 and H a r r i s 1988). The re fo re , the i n t e r f e r e n c e w i th the C a - a c t i v a t e d K-conductance mechanism of the AHP i n n e o c o r t i c a l neurons t h a t was produced by i s o f l u r a n e or A l t h e s i n a l so cou ld be a d i r e c t r e s u l t of decreased inward Ca-cur rent s ( K r n j e v i c ' a n d P u i l 1988), or may be r e l a t e d t o l e s s s p e c i f i c anaes the t i c a l t e r a t i o n s i n the 2+ / s t e ady - s t a t e background of [Ca ] . ( c f . K r n j e v i c 1974a; M o r r i s 1986). 7.2.2 S i g n i f i c a n c e of AHP i n h i b i t i o n i n n e o c o r t i c a l neurons. The importance of the s low AHPs i n s u b c o r t i c a l neurons ( B a l d i s e r r a and Gustafsson 1974; K r n j e v i c e t a l . 1978b) and the medium-duration AHPs i n n e o c o r t i c a l neurons (Schwindt et a l . 1988a,b) i s w e l l app rec i a ted f o r r e g u l a t i n g neuronal h y p e r e x c i t a b i l i t y . Ca r l en e t a l . (1985) observed that sedat i ve s i nc reased the AHPs and reduced r e p e t i t i v e f i r i n g i n va r ious s u b c o r t i c a l neurons. The blockade of the slow AHP gene ra t i on i n such neurons by apamin (Zang and K r n j e v i c ' 1987) or i n aged hippocampal neurons by ethanol (Niesen e t a l . 1988), i nc rea se s r e p e t i t i v e sp i ke d i s cha rge . In neocortex, a depres s ion of r e p e t i t i v e f i r i n g a b i l i t y accompanied the anae s the t i c b lockade of the medium-duration AHP. The apparent paradox may be r e l a t e d to the obse rva t ions t h a t we l l - de ve l oped , f a s t AHPs i n hippocampal neurons are not reduced by a p p l i c a t i o n s of apamin or ethanol (Zhang and H. EL-BEHEIRY 138 K r n j e v i c 1987; Niesen et a l . 1988). Such AHPs are not mediated by C a 2 + and are not prominent i n neooco r t i c a l neurons (Schwindt e t a l . 1988a,b). The anae s the t i c depress ion of r e p e t i t i v e f i r i n g i n neocortex cou ld be a consequence of a removal of the major mechanism by which Na-channels are primed f o r a c t i v a t i o n . For example, depress ion of the medium-duration AHPs i n neocortex by anaes thes ia would prevent the l a t e r e p o l a r i z i n g s h i f t of the membrane p o t e n t i a l which normal ly decreases N a - i n a c t i v a t i o n and re se t s the e x c i t a t i o n c y c l e f o r N a - a c t i v a t i o n , r e s u l t i n g i n an a t t e n u a t i o n of r e p e t i t i v e sp ike d i s cha rge . 8 EFFECTS OF ISOFLURANE ON THE GLUTAMATE- AND POTASSIUM-INDUCED  INCREASE IN INTRANEURONAL CALCIUM CONCENTRATION The o b j e c t i v e s of t h i s s e r i e s of i n v e s t i g a t i o n s were: (1) to e l u c i d a t e the mechanisms of the depressant a c t i o n s of anae s the t i c s on the ACh- and Glu-evoked d e p o l a r i z a t i o n ( c f . S e c t i o n 5 ) , 2+ (2) t o monitor p o s s i b l e changes i n the r e s t i n g l e v e l of [Ca ] . induced by i s o f l u r a n e , and (3) to study the e f f e c t s of i s o f l u r a n e on the p re - and pos t synapt i c a c t i v a t i o n of vo l tage dependent Ca-channels . Cu l t u r ed f e t a l hippocampal neurons were chosen f o r these i n v e s t i g a t i o n s p a r t l y because they represent a we l l c h a r a c t e r i z e d model f o r the study of 2+ the Ca homeostasis i n the c e n t r a l nervous system ( c f . Kudo and Ogura 1986). 8.1 Re su l t s Re s t i ng [ C a 2 * ] ^ l e v e l s were 63.8 (± 38.9) nM (n = 30 ) . [ C a 2 + ] 1 were measured f o r ~2 min p r i o r to agon i s t o r drug a p p l i c a t i o n ; neurons w i th H. EL-BEHEIRY 139 r e s t i n g [Ca 2 * ] . . va lues above 150 nM or those w i t h r e s t i n g [ C a 2 + ] . t h a t f l u c t u a t e d more than ±20 nM were not i n c l uded f o r a n a l y s i s . [These c o n d i t i o n s occurred r a r e l y and seemed to be r e l a t e d to poor c e l l v i a b i l i t y . ] S i m i l a r volumes of c o n t r o l s o l u t i o n were i n j e c t e d on d i f f e r e n t occas ions t o t e s t f o r i n j e c t i o n ( p o s s i b l y mechanica l ) a r t i f a c t s . 8.1.1 Response to ACh and c a r b a c h o l . Small i nc rea se s (~25 nM) i n 2+ [Ca ] . were observed (3/12 neurons) when ACh or carbacho l i n the micromolar range (10-200 nM) was app l i ed i n small volumes (25 ul) by i n j e c t i o n as a bolus i n t o the reco rd ing chamber. The evoked responses had a l a tency of ~20s and a du ra t i on of 100-150s. Th i s i s c o n s i s t e n t w i th the obse rva t i on s of Benavides and a s s o c i a t e s (1988; c f . Kudo e t a l . 1988) i n b r a i n synaptoneurosomes. The e f f e c t s of i s o f l u r a n e cou ld not be w e l l 2+ s t ud i ed because the increments i n [Ca ] . evoked by the c h o l i n e r g i c agon i s t s were too small i n t h i s model. 8.1.2 E f f e c t s of i s o f l u r a n e on r e s t i n g [Ca 2 * ] . j and Glu a c t i o n s . Continuous pe r f u s i on of media s a tu ra ted w i t h 1.5 and 2.5 MAC i s o f l u r a n e 2+ produced only small and i n c o n s i s t e n t changes i n the r e s t i n g [Ca ] . over a pe r i od of 20 min. A p p l i c a t i o n of 50 LIM Glu (25 Ltl volumes) to 18 neurons 2+ evoked a mean inc rease of 220 ±78 nM i n [Ca ] . . The responses were instantaneous and had an " on/o f f " type of p a t t e r n ( F i g . 53) . When TTX was added to the per fu s ing s o l u t i o n , s i m i l a r responses were obta ined w i t h 50 uM Glu a p p l i c a t i o n . 2+ Pe r f u s i on of i s o f l u r a n e 0.5 MAC had no e f f e c t s on the [Ca ] . i nc reases evoked by Glu (4/8 neurons) or p o t e n t i a t e d the Glu responses ( F i g . 53) i n TTX f r e e media. A t t e n u a t i o n of the Glu responses i n media f a vou r i n g NMDA or Quis was always observed on a p p l i c a t i o n of 1.75 and 2.5 MAC i s o f l u r a n e ( F i g s . 54 and 55, Table 3 ) . I s o f l u r ane 2.5 MAC H. EL-BEHEIRY 140 F i g . 53 P o t e n t i a t i o n of responses to glutamate i n j e c t i o n s (25 uM; • ) i n 5 c o n s e c u t i v e l y sampled neurons by 0.5 MAC i s o f l u r a n e under QUIS c o n d i t i o n s . H. EL-BEHEIRY 141 300 200 CM + CO o — 100 0 0 • IFL 1.75 MAC dD ftCbg *-qJ b - ^ 3\ a h 2 0 4 0 Time Imin) 6 0 Fig. 54 Isoflurane (1.75 MAC) inhibit ion of increases in [Ca 2 + ] j produced by glutamate application (50 uM; A ) under NMDA conditions. H. EL-BEHEIRY 142 600. A A A A A A A A A A A A H 1 1 1 1 1 1 1 r-0 20 40 60 80 Time (min 1 F i g . 55 I s o f l u r ane (2.5 MAC) blockade o f i n c rea se i n [ C a 2 + ] i by glutamate a p p l i c a t i o n (50 U M; • ) under Quis c o n d i t i o n s . H. EL-BEHEIRY 143 Table 3 E f f e c t s of i s o f l u r a n e on [Ca ] . i nc reases evoked by g lutamate and K + . Increases i n [Ca 2 +]-{ Depress ion*by i s o f l u r a n e (MAC) evoked by 0.5 1.5 1.75 2.5 NMDA {TTX present) 64 ± 14.4% (n = 4) (No TTX) No e f f e c t * * 60 * 10% (n = 3) (n = 8) QUIS (TTX present) 52 ± 12.4% 85 * 13.4% (n = 8) (n = 4) (No TTX) 41 ± 15.3% (n = 4) K + (TTX present) 45 ± 21% (n = 6) * Depress ion was de f ined as >20% decrease i n the responses induced by g lutamate or K + i n j e c t i o n s than i n c o n t r o l c o n d i t i o n s . P o t e n t i a t i o n of g lutamate responses was observed i n 5 neurons. H. EL-BEHEIRY 144 complete ly suppressed the Glu responses such t h a t on ly p a r t i a l recovery was observed w i t h i n the exper imenta l pe r i od ( F i g . 55) . These ob se r va t i on s were c o n s i s t e n t de sp i t e the presence or absence of TTX. I n h i b i t i o n of the Glu responses requ i red 6 min of anaes the t i c a p p l i c a t i o n . Recovery was observed a f t e r 8-12 min. 8.1.3 I s o f l u rane a c t i on s on K-evoked inc reases i n [Ca ] . . K (50 mM) s lowly i n j e c t e d as a 25 u l bolus i n t o the r eco rd i ng chamber i nc rea sed [ C a 2 + ] i by 300 ±95 nM (n = 15) i n the absence of TTX ( F i g . 2+ 56) . In the presence of TTX the increments of [Ca ] . were s l i g h t l y g r ea te r i n 7 neurons (Table 3 ) . I s o f l u r ane (1.5 MAC) a d m i n i s t r a t i o n depressed the i n c rea se i n 2+ + [Ca ].j evoked by K bo lus i n j e c t i o n s by 45 * 22% i n the absence of TTX. S i m i l a r depress ions were observed i n the presence of the Na-channel b l o c ke r i n 4 neurons. 8.2 D i s cu s s i on The f a i l u r e of i s o f l u r a n e a p p l i c a t i o n a t h igh doses (2.5 MAC) to 2+ c o n s i s t e n t l y i nc rease basa l [Ca ] . i s not i n agreement w i t h o ther ob se rva t i on s i n r a t thymocyte c e l l suspensions (Mor r i s 1986) and b r a i n synaptosomes (Daniel1 and Ha r r i s 1988). D i f f e r e n t models used may account f o r the d i s c repan t r e s u l t s . A drawback of synaptoneurosomes, compared w i t h c u l t u r e d neurons, i s t h e i r poorer s u r v i v a l s t a b i l i t y and probable he te ro -gene i ty i n s i z e as we l l as r e cep to r s . In a d d i t i o n they possess s i g n i f i c a n t l y 2+ h i gher l e v e l s of r e s t i n g [Ca ] . than those reported f o r i n t a c t c e l l s ( c f . Benavides et a l . 1988). In the present i n v e s t i g a t i o n s , Glu responses were evoked i n the presence o r absence o f 800 uM Mg i n the bath ing s o l u t i o n . This c o n c e n t r a t i o n i s s u f f i c i e n t f o r blockade of the NMDA-type of r ecepto r s a c t i v a t e d by Glu (Nowak H. EL-BEHEIRY 145 F i g . 56 Vo l tage-gated i nc rea se i n [ C a 2 + ] i produced by K + (50 and 100 LIM, • ) bo lus i n j e c t i o n s t h a t were depressed by i s o f l u r a n e a d m i n i s t r a t i o n under Quis c o n d i t i o n s . The anaes the t i c depress ion was observed i n the presence of TTX. Recovery was observed w i th the h i ghes t bo lus of K + (100 nM). H. EL-BEHEIRY 146 et a l . 1984). Consequently, the responses recorded were due main ly to a c t i v a t i o n of e i t h e r the NMDA or the Qu i s - t ype of r e cep to r s (Murphy e t a l . 1987). The a d d i t i o n of TTX would have ensured an i s o l a t i o n of the neurons from impinging s ynapt i c i npu t s . An a t t enua t i on of the G lu - induced r i s e i n 2+ [Ca ] . has been repor ted p r e v i ou s l y f o r i s o f l u r a n e as w e l l as halothane ( P u i l and Baimbridge 1989). However, the mechanisms of i n t e r f e r e n c e of anae s the t i c s w i th the Glu ac t i on s are not we l l understood. G l u , by a c t i v a -t i n g the Qu i s - subtype of r e cep to r s , induces two d i s t i n c t responses i n i n v i t r o hippocampal neurons (Murphy and M i l l e r 1989; c f . Furuya e t a l 2+ 1989.). A t r a n s i e n t b r i e f increase i n [Ca ] . superimposed on top of a more mainta ined p l a teau response. The prolonged response was abo l i s hed by 2+ + removal of Ca o r Na from the ex te rna l media or by b l o ck i n g vo l t age -s e n s i t i v e Ca-channels (Murphy and M i l l e r 1989). In c o n t r a s t , none of these 2+ man ipu la t i on s abo l i s hed the t r a n s i e n t [Ca ] . s p i k e . A c c o r d i n g l y , the prolonged response has been suggested to r e s u l t from a c t i v a t e d v o l t a ge -2+ dependent Ca-channels whereas the t r a n s i e n t [Ca ] . sp ike r e s u l t e d from 2+ Ca m o b i l i z a t i o n from i n t r a c e l l u l a r s t o re s which i n t u rn i s due to agon i s t - i nduced i n o s i t o l t r i pho spha te (IP^) s yn the s i s (Ber r idge 1987; N i c o l e t t i e t a l . 1988), mediated by phosphol ipase C e f f e c t s at the receptor/G p r o t e i n l e v e l (Katada et a l . 1985; E l -Fakahany e t a l . 1988). 2+ The a c t i o n s of i s o f l u r a n e on the Glu-evoked i nc rea se s i n [Ca ] . most l i k e l y i n v o l v e blockade of voltage-dependent Ca-channels ( c f . Lodge and Shaughnessy 1988; K rn j e v i c ' and P u i l 1988) and/or d i r e c t i n t e r f e r e n c e w i th the coup l i n g of i o n i c channels t r i g g e r e d by the i n t e r a c t i o n s of Glu w i th i t s r e cep to r s . 2+ + Increments of [Ca ] . evoked by K a p p l i c a t i o n s were depressed by i s o f l u r a n e de sp i t e the presence or absence of TTX. Th i s p rov ides presumptive H. EL-BEHEIRY 147 ev idence t h a t anae s the t i c s a f f e c t voltage-dependent Ca-channels at p r e - and po s t s ynap t i c s i t e s . Hence, the observed depress ion of EPSPs and IPSPs ( c f . S e c t i o n 4) cou ld r e s u l t from decreased t r a n s m i t t e r r e l ea se and a t t e n u a t i o n of po s t s ynap t i c s e n s i t i v i t i e s to t r a n s m i t t e r s . 9 GENERAL DISCUSSION These i n v e s t i g a t i o n s p r o v i de , f o r the f i r s t t ime , comprehensive observa-t i o n s main ly on the e f f e c t s of i s o f l u r a n e and A l t h e s i n i n n e o c o r t i c a l neurons which are l i k e l y t a r ge t s i t e s i n the p roduct ion of the anaes the t i c s t a t e . The n e o c o r t i c a l i n v i t r o s l i c e p r epa r a t i on a l lowed w e l l - c o n t r o l l e d exper iments to be c a r r i e d out , de sp i t e c e r t a i n gene ra l i z ed r e s e r v a t i o n s about the i n v i t r o s l i c e p r e p a r a t i o n , e . g . , q u a l i t a t i v e and q u a n t i t a t i v e aspects of metabolism t h a t may be d i f f e r e n t from tha t i n the i n v i vo s i t u a t i o n ( c f . Kovachich and M i shra 1981), removal of the t o n i c i n f l u e n c e o f ascending arousa l pathways and u n c e r t a i n t i e s about the achievement of opt imal drug l e v e l s . For example, some of the pharmacokinet ic parameters t h a t comp l i ca te the i n t e r p r e t a t i o n of r e s u l t s obta ined from s tud ie s i n the i n t a c t o r decerebrate animal ( i n v i vo p repa ra t i on ) may be minimized i n the s l i c e p r e p a r a t i o n . Because n e o c o r t i c a l s l i c e s are devoid of i n f l u e n c e s from e x t r a c o r t i c a l a f f e r e n t a c t i v i t y , the anae s the t i c a c t i on s are a t t r i b u t a b l e most ly t o the neuron under i n v e s t i g a t i o n , p a r t i c u l a r l y when TTX a p p l i c a t i o n had been employed to reduce a c t i v i t y emanating from the i n t r a c o r t i c a l connec t i on s . In a d d i t i o n * the i n v i t r o s i t u a t i o n a l lowed man ipu l a t i on of the i o n i c environment f o r the e x p l o r a t i o n of the mechanisms of d r ug - a c t i on s and f o r s imu l a t i on s of va r i ous p a t h o p h y s i o l o g i c a l c o n d i t i o n s ( e . g . , hypo-magnesaemia). Chemical dete rminat ions of drug concen t ra t i on s in the bath ing medium ( c f . Methods) assured the presence of opt imal l e v e l s i n the s l i c e , e s p e c i a l l y f o r dose-response s t u d i e s . H. EL-BEHEIRY 148 9.1 Neoco r t i c a l u n i t a c t i v i t y and general anaesthes ia S u b s t a n t i a l ev idence has been prov ided by many i n v e s t i g a t o r s t h a t the spontaneous a c t i v i t y of n e o c o r t i c a l neurons, depending on t h e i r exact l o c a t i o n and animal spec ie s i n v e s t i g a t e d , changes w i t h time and accord ing t o the a n i m a l ' s degree of a l e r t n e s s (Eva r t s 1964; Noda and Adey 1970; Webb 1976 a,b; F o s t e r e t a l . 1982). Moreover, Noda and Adey (1973) were not ab le to de t e c t any d i f f e r e n c e s between the e f f e c t s of t h i o p e n t a l anaes thes ia and normal s leep on the temporal p a t t e r n of d i scharge from s i n g l e neurons i n the c a t a s s o c i a t i o n a l c o r t e x . Such modulat ion of n e o c o r t i c a l sp ike a c t i v i t y dur ing anaesthes ia has been a t t r i b u t e d to anaes thet i c a c t i o n s on the a f f e r e n t pathways (Mountcast le e t a l . 1957; M e r l i s 1965; Ber ry and Hance 1965; Greenbaum and M e r l i s 1965). However, Robson (1967) repor ted t ha t anae s t he t i c s un i fo rmly attenuated the a c t i v i t i e s of spontaneously f i r i n g n e o c o r t i c a l un i t s i n decerebrate an imals . The present i n v e s t i g a t i o n s of s l i c e p repa ra t i on s revea led t h a t anae s the t i c - i nduced depress ion of the spontaneous a c t i v i t y i s due to d i r e c t a c t i on s on the c e r e b r a l c o r t i c a l neurons. The reduc t i on i n spontaneous a c t i v i t y and the i n a b i l i t i e s of the neurons to f i r e sp i kes r e p e t i t i v e l y would tend to d i s r u p t the genera t i on of meaningful s p i k i ng pa t t e rn s and g e n e r a l l y impede a t r a n s c o r t i c a l spread o f i n f o r m a t i o n . A c c o r d i n g l y , the p r e v a i l i n g l e v e l of c e r eb r a l c o r t i c a l a c t i v a -t i o n and generat ion of memory t r a c e s would be reduced to the extent t h a t c o r t i c a l pe rcept i on of the i n t e r n a l and ex te rna l environments would be obtunded, r e s u l t i n g i n an anae s the t i c (unconscious) s t a t e . 9.2 Anaes thet i c a c t i o n s : depress ion of e x c i t a t i o n or p o t e n t i a t i o n of  i n h i b i t i o n ? The observed s e l e c t i v e i n t e r f e r e n c e w i t h the a c t i on s of a c t i v a t i n g t r a n s m i t t e r s and c o r t i c a l EPSPs observed i n the present s t ud i e s support the H. EL-BEHEIRY 149 concept t h a t anae s the t i c a c t i o n s , at l e a s t i n the neocor tex , are due to depress ion of e x c i t a t i o n and not a r e s u l t of p o t e n t i a t i o n of i n h i b i t i o n . Enhancement o r p r o l onga t i on of i n h i b i t o r y processes i n the b r a i n i s a l i k e l y mechanism of a c t i o n of c e r t a i n s opo ro f i c agents, e . g . , benzod iazep ines or b a r b i t u r a t e s i n sedat i ve doses (Car len et a l . 1985). On the other hand, the s t r i c t i n t r i n s i c and e x t r i n s i c c o n t r o l of e x c i t a t o r y processes p rov ides a d i r e c t and s t r i n g e n t r e g u l a t i o n of neuronal e x c i t a b i l i t y i n the b r a i n . N e o c o r t i c a l neurons r e c e i v e sus ta ined a c t i v a t i o n from i n t r a c o r t i c a l synapt ic inputs ( c f . S e c t i o n 3) as we l l as from ascending pathways, p a r t i c u l a r l y those of the RAS (Matsumura e t a l . 1988). Hence, an a t t e n u a t i o n of e x c i t a t i o n p a r t i c u l a r l y in the neocortex prov ides a p l a u s i b l e c e l l u l a r ba s i s f o r the anae s the t i c s t a t e . Th i s i s not easy to r e c o n c i l e w i t h the n o n - s p e c i f i c l i p i d hypothes i s t h a t p r e d i c t s a d i s s o l u t i o n of anae s t he t i c molecu les i n the l i p i d s of the plasma membrane which produces anaes thes i a . The present r e s u l t s , however, are c o n s i s t e n t w i th s p e c i f i c i n t e r a c t i o n s of anae s the t i c s w i t h hydrophobic p r o t e i n s (Franks and L i e b , 1987). The depress ion of the i n h i b i t o r y responses to GABA demonstrated i n n e o c o r t i c a l neurons may u n d e r l i e the e x c i t a t o r y phenomena t h a t r epo r t ed l y accompany i nduc t i on of anaes thes ia w i th A l t h e s i n or i s o f l u r a n e ; a l o g i c a l p revent i ve measure would be the premedicat ion w i t h a GABA-potent ia t ing agent such as a benzodiazepine (V i cke r s et a l . 1981; R i c h t e r 1981). 9.3 The anae s the t i c s t a t e and suppress ion of consc iousness mechanisms The processes generat ing consciousness i n the c e r e b r a l c o r t ex are dependent on a high degree of spontaneous and r e p e t i t i v e a c t i v i t y ( L i b e t 1965; Webb 1983) which i s promoted by the a c t i o n s of ACh (Woody e t a l . 1978) l i b e r a t e d onto the neurons as a consequence of the su s ta ined a c t i v i t y of the ascending RAS (Szerb 1967; K r n j e v i c 1969; Shute and Lewis 1963; Kimura e t H. EL-BEHEIRY 150 a l . 1981). ACh markedly enhances c o r t i c a l responses to e l e c t r i c a l s t i m u l a -t i o n and s y n a p t i c a l l y mediated d e p o l a r i z a t i o n ( K r n j e v i c e t a l . 1971b; Dodd et a l . 1981). The f a c i l i t a t o r y a c t i on of ACh at musca r i n i c recepto r s i s slow to develop and con fe r s a g reate r tendency of the neurons to d i scharge r e p e t i t i v e l y ( K r n j e v i c 1981, 1989; McLennan 1983). Consequently, the c h o l i n e r g i c RAS t o n i c a l l y r a i s e s the o v e r a l l l e v e l of c o r t i c a l a c t i v i t y and enhances the neuronal respons iveness to " on/o f f " t r a n s m i t t e r s l i k e Glu f o r a f a s t , s p e c i f i c t r a n s f e r of i n fo rmat ion ( P u i l and Benjamin 1988) between neurons i n the consc ious s t a t e . These ac t i on s are probably e s s e n t i a l f o r the maintenance of a l e r t n e s s and c o n t r i b u t e t o the content of consc iousness . A decrease i n the background l e v e l of c o r t i c a l a c t i v i t y can " t une " the consc ious system t o lower degrees of a l e r t n e s s and a r o u s a l . Hence, a complete suppress ion of the ascending c h o l i n e r g i c d r i v e would obtund c o r t i c a l pe r cep t i on of the sensory m o d a l i t i e s a r i s i n g from s u b c o r t i c a l a c t i v i t i e s and r e s u l t i n an unconscious s t a t e ( c f . F rancescon i et a l . 1988). Anae s t he t i c s depress t h i s tone e i t h e r by b l o c k i n g the i n t r i n s i c l e v e l of e x c i t a b i l i t y of the RAS o r by a t t enua t i n g the responses of n e o c o r t i c a l neurons to ACh s y n a p t i c a l l y re leased by the cont inuous e x c i t a n t a c t i on s of the r e t i c u l a r f o rmat ion . The r e s u l t s of French et a l . (1953; c f . a l s o Morruz i and Magoun 1949) and others (Haugen and Melzack 1957; Davis et a l . 1957) favoured the concept t h a t anae s the t i c s i n t e r f e r e w i t h the r e t i c u l a r fo rmat ion i n the process ing of sensory inputs to the c o r t e x . The f i nd i n g s were i n t e r p r e t e d as being due t o the g rea te r number of synapt ic connect ions t h a t , accord ing to Barany (1947), would render the RAS more vu lne rab le to a nae s t he t i c a c t i on s than any o ther areas of the b r a i n . Th i s t a c i t assumption has remained the most w ide ly accepted view of the e s s e n t i a l c e n t r a l a c t i on s of a n a e s t h e t i c s , de sp i t e the H. EL-BEHEIRY 151 r e l a t i v e p a u c i t y of c r i t i c a l ev idence. Severa l papers have repor ted t h a t v o l a t i l e and int ravenous agents e q u a l l y depress po l y - as we l l as monosynaptic pathways i n d i f f e r e n t areas of the neurax i s (Au s t i n and Pask 1952; E s p l i n 1963; De Jong e t a l . 1968). Recent b iochemica l f i n d i n g s a l so i n d i c a t e t ha t the RAS i s not a primary s i t e of depressant a c t i o n of a n a e s t h e t i c s , whereas a g ene r a l i z ed or d i f f u s e depress ion of the c e r e b r a l co r tex i s c o n s i s t e n t l y observed ( c f . I n t r o d u c t i o n ) . The major hypothes i s developed dur ing the course of these i n v e s t i g a t i o n s suggests t h a t general anae s the t i c s depress chemica l s e n s i t i v i t i e s o f n e o c o r t i c a l neurons to s y n a p t i c a l l y re lea sed a c t i v a t i n g t r a n s m i t t e r s thereby a t t e n u a t i n g the sus ta ined tone of the RAS and r e - s e t t i n g the c o r t i c a l a rousa l system to a lower steady s t a t e l e v e l of a c t i v i t y which c o n s t i t u t e s " the anae s t he t i c s t a t e " . P a r t i a l v e r i f i c a t i o n of the hypothes i s was achieved i n the dose-response s t ud i e s which showed t h a t both i s o f l u r a n e and A l t h e s i n s e l e c t i v e l y depressed the d e p o l a r i z a t i o n s evoked by e x t r a c e l l u l a r a p p l i c a -t i o n s of ACh i n anaes thet i c concen t r a t i on s cor respond ing to those observed i n the b r a i n of anaes the t i zed an imals . The a t t e n u a t i o n of the f a s t responses induced by Glu produced lower s lopes i n the dose-response curves f o r both agents than the s lopes of the dose-response curves c h a r a c t e r i z i n g the dose-dependent anaes the t i c i n t e r f e r e n c e w i t h the e x c i t a t o r y a c t i o n s of ACh. Hence, both ACh- and Glu-evoked responses are depressed at a given dose w i t h vary ing degrees of anaes the t i c e f f i c a c y . By app ly ing t h i s i n t e r p r e t a t i o n to the i n v i v o s i t u a t i o n , s u r g i c a l l e v e l s of anaes thes i a would be expressed when the t o n i c e x c i t a t i o n produced by ACh i n the c e r e b r a l co r tex i s depressed whereas the f a s t e r a c t i on s of Glu are on ly s l i g h t l y a f f e c t e d . Deeper l e v e l s of the anae s the t i c s t a t e would r e f l e c t i n t e r f e r e n c e w i t h the a c t i o n s of both a c t i v a t i n g t r a n s m i t t e r s . H. EL-BEHEIRY 152 The above observat ions are c o n s i s t e n t w i th the recent c o n c e p t u a l i z a t i o n of the anae s the t i c s t a t e as a cumulat ive continuum i . e . , a s e r i e s of t r an s f o rma t i on s pass ing i n t o each o the r , s t a r t i n g w i th ana l ge s i a and emerging i n t o amnesia and l o s s of n e u r o l o g i c a l respons iveness (White 1987). In order t o exp lo re the v a l i d i t y of the hypothes i s , the consc ious s t a t e was s imu la ted by an i n v i t r o neuronal model of neocortex and the e f f e c t s of the anaesthe-t i c s was i n v e s t i g a t e d t h e r e i n . Both i s o f l u r a n e and A l t h e s i n complete ly b locked the e x c i t a t i o n sus ta ined by a cont inuous ACh a p p l i c a t i o n w i th a low c u r r e n t i n t e n s i t y ( c f . F i g . 37 and 38) . During t h i s t ime , the superimposed b r i e f G l u - d e p o l a r i z a t i o n s evoked a t c o n s i s t e n t i n t e r v a l s were s l i g h t l y depressed. Because these s t r u c t u r a l l y d i s s i m i l a r anae s the t i c s produced nea r l y i d e n t i c a l e f f e c t s i n t h i s neuronal model, the f o l l o w i n g suggest ions may be r e l e v a n t t o the mechanism of the anaes the t i c s t a t e . That i s , dur ing anae s the s i a , the re are pha rmaco log i ca l l y s p e c i f i c a l t e r a t i o n s of the su s ta i ned ascending c h o l i n e r g i c i npu t i n neurons w i t h i n the c e r e b r a l co r tex which r e se t s t h e i r r e s p o n s i v i t i e s to the s y n a p t i c a l l y r e l ea sed t r a n s m i t t e r s . Th i s modulat ion may be l a r g e l y con t i ngent on the a b i l i t i e s of anae s the t i c s to modulate the respons iveness of the po s t s ynap t i c recepto r s of n e o c o r t i c a l neurons. 9.4 I on i c mechanisms of anae s the t i c a c t i o n The e f f e c t s of i s o f l u r a n e and A l t h e s i n on n e o c o r t i c a l neurons i n these i n v e s t i g a t i o n s can be summarized accord ing to f i v e bas i c ob se r va t i on s : (1) depress ion of spontaneous a c t i v i t i e s of the neurons, (2) a t t enua t i on of evoked e x c i t a t o r y and i n h i b i t o r y s ynap t i c t r a n s i e n t s , (3) suppress ion of the "chemica l respons iveness " t o the e x t r a c e l l u l a r a p p l i c a t i o n of ACh, G lu , NMDA and GABA near the somata, H. EL-BEHEIRY 153 (4) a t t enua t i on of the " pha rmaco l og i c a l l y i s o l a t e d " C a - a c t i v a t e d K-conductance ( i . e . AHP genes i s ) and (5) blockade of Glu and K-evoked inc reases i n the [Ca ] . . The most pars imonious exp l ana t i on f o r these e f f e c t s i s t h a t the anaes-t h e t i c agents i n t e r f e r e d w i t h C a - i n f l u x through vo l tage-dependent and recepto r -opera ted Ca-channels at p r e - and pos t s ynapt i c s i t e s . Consequent ly, t r a n s m i t t e r re lease and slow inward pos t synapt i c Ca - cu r ren t s r e spon s i b l e f o r the r e p e t i t i v e s p i k i n g a c t i v i t i e s of the neurons are decreased ( c f . S ta f s t rom e t a l . 1985) whereas the Ca-dependent responses to ACh, G l u , NMDA and GABA would be attenuated ( c f . Inoui e t a l 1986; M i sge ld et a l . 1986; S c h o l f i e l d e t a l . 1987; Gahwi ler and Brown 1987; Murphy and M i l l e r 1988; Benavides e t a l . 1988; Bormann 1988, Akaike 1990). The dose-dependent suppress ion o f the i s o l a t e d C a - a c t i v a t e d K-conductance ( c f . S ec t i on 5) together w i th the a t t enua t i on of Ca-sp ikes (Heyer and Macdonald 1982) and Ca -cur rent s (N i s h i and Oyama 1983; K r n j e v i c and P u i l 1988; Gross and Macdonald 1988; Gunderson e t a l 1988) suggest t h a t voltage-dependent Ca-conductances are s i g n i f i c a n t l y reduced by the anaes the t i c agents. Furthermore, i t may be suggested t h a t an anae s t he t i c - i nduced increase i n [Ca ] . (K rn jev i c * 1974b; 1986), may lead to i n a c t i v a t i o n of Ca-channels ( c f . E c k e r t and Chad 1984). Th i s would be expected on the ba s i s t ha t anae s the t i c s s imu la te the ac t i on s of metabo l i c i n h i b i t o r s (Quastel 1952, 1962; Godf ra ind e t a l . 1971; Krn jev ic * e t a l 1978a; Vanderkooi et a l . 1981) and mimick the e f f e c t s of r e v e r s i b l e hypoxia ( c f . Fu j iwa ra e t a l . 1987; Lablond and K r n j e v i c 1989; K rn jev i c ' and Walz 1990). Such an assumption i s d i f f i c u l t t o r e c o n c i l e w i th the present i n v e s t i g a t i o n s ( c f . S e c t i o n 6) as we l l as w i th recent r epo r t s 2+ t h a t a s i g n i f i c a n t i n c rea se i n [Ca ] . was not observed dur ing a p p l i c a t i o n s of anae s t he t i c s ( D a n i e l l and Ha r r i s 1988; P u i l and Baimbridge 1989). H. EL-BEHEIRY 154 Another p o s s i b i l i t y i s t h a t anaes thet i c molecules b ind to a c y t o s o l i c p r o t e i n i n vo l ved i n secondary messenger t r a n s d u c t i o n processes ( c f . F i r e s t o n e 1988). One such candidate i s p r o t e i n k inase C (PKC). PKC i s a C a - a c t i v a t e d phosphol ip id-dependent enzyme t ha t phosphory lates a v a r i e t y of c e l l u l a r p r o t e i n s i n vo l ved i n transmembrane s i g n a l i n g . The a c t i v i t y of t h i s enzyme has been f i r m l y l i n k e d to s i gna l t r a n s d u c t i o n by the demonstrat ions t h a t d i a c y l g l y c e r o l , one of the i n i t i a l products of musca r i n i c and g lu tamate rg i c r e c e p t o r - a c t i v a t e d i n o s i t o l phospho l i p id h y d r o l y s i s (Ber r idge 1987; P e r a l t a e t a l . 1988; Sladeczek e t a l . 1988), g r e a t l y i nc reases the a f f i n i t y of PKC f o r Ca, thereby causing i t s a c t i v a t i o n . In the b r a i n t h i s enzyme which i s most ly a s s oc i a ted wi th synapses (Kikkawa e t a l . 1982) a f f e c t s recepto r s e n s i t i v i t y (DeRiemer et a l . 1985) and ion channel a c t i v i t y (Castagna et a l . 1982; Conn e t a l . 1989). Moreover, F i r e s t o n e and F i r e s t o n e (1988) demonstrated tha t anaes thet i c s can i n h i b i t P K C - a c t i v i t y i n i s o l a t e d r a t b r a i n homogenates. A s i m p l i f i e d r ep re sen t a t i on of the sequence of events may be schematized as below: Anae s t he t i c s b ind w i t h and i n h i b i t the a c t i v i t y of p r o t e i n k inase C. Voltage-dependent Ca-channels are no longer e f f i c i e n t l y phosphory lated and hence i n a c t i v a t e d . Glu (Sugiyama et a l . 1987; S ladeczek e t a l . 1988) and NMDA (Ber r idge 1987) s t imu l a te the phospho ino s i t i de (PI) h y d r o l y s i s c y c l e thereby i n c r ea s i n g i n t r a c e l l u l a r Ca - concen t ra t i on which a c t i v a t e s p r o t e i n k inase C to phosphory late the recepto r -opera ted channe l s . However, the p r o t e i n k inase C a l ready would be i n h i b i t e d by the anae s the t i c b i nd i ng . Therefore the i n o s i t o l phosphate d i a c y l g l y c e r o l second messenger pathway t h a t t r i g g e r s a p o s i t i v e feedback mechanism f o r a f u r t h e r i nc rease i n C a - i n f l u x would be b l ocked . Musca r i n i c receptor s f o r ACh i n the b r a i n are known to a c t i v a t e P i - h y d r o l y s i s (Malenka et a l . 1986; P e r a l t a e t a l . 1988; H. EL-BEHEIRY 155 Dutar and N ico l1 1988) and t h e i r e f f e c t s are thought to be mediated through a p r o t e i n k inase i n n e o c o r t i c a l neurons (Woody and Gruen 1988; c f . El-Fakahany e t a l . 1989). Despite a lack of understanding of the l i n k between the i o n i c ba s i s of the muscar in i c ACh induced responses and the secondary messenger systems, the presumed i n h i b i t i o n of the PKC by anae s the t i c agents can e x p l a i n the depress ion of ACh-evoked d e p o l a r i z a t i o n s dur ing anae s the s i a . In a d d i t i o n , t h e i r e f f e c t s on GABA recepto r s are probably mediated through a p r o t e i n k inase enzyme. Indeed the phosphory la-t i o n s i t e on the cy top la smic reg ion of the GABA^ recepto r has been i d e n t i f i e d ( S c h o l f i e l d e t a l . 1987; S i g e l and Baur 1988; Gyenes e t a l . 1988). An anaes thet i c induced i n h i b i t i o n of PKC may be p a r t l y re spons ib le f o r the depress ion of the GABA-induced d e p o l a r i z a t i o n s observed i n these i n v e s t i g a t i o n s . 9.5 Mo lecu l a r mechanisms of anaesthes ia - - the l i p i d / p r o t e i n controversy Because of the d i f f i c u l t y i n r e c o n c i l i n g d i s s i m i l a r i t i e s i n chemical s t r u c t u r e s of anae s the t i c agents with convent iona l ' l o c k and key ' concepts of d rug - recepto r i n t e r a c t i o n s , a n a e s t h e s i o l o g i s t s have not favoured s p e c i f i c or p r o t e i n models of anaes thes i a . In s tead, n o n - s p e c i f i c or l i p i d - b a s e d models, have enjoyed most of the exper imenta l a t t e n t i o n ( c f . J ano f f 1982; F i r e s t o n e and K i t z 1988; F i r e s t one 1988). To some ex ten t , the present i n v e s t i g a t i o n s cha l l enge the assumptions suggest ing t ha t the important a c t i on s of anae s the t i c s are induced by n o n - s p e c i f i c b i o p h y s i c a l a l t e r a t i o n s of membrane p r o p e r t i e s . Moreover, the s i m i l a r r e s u l t s obta ined using c h e m i c a l l y d i s t i n c t agents ( i s o f l u r a n e and A l t h e s i n ) i n d i c a t e t h a t anaesthe-t i c s c ou l d a c t s p e c i f i c a l l y a t s i m i l a r hydrophobic s i t e s r a t he r than b ind ing to d i f f e r e n t p r o t e i n pockets as suggested e a r l i e r ( c f . R ichards 1975; 1983). H. EL-BEHEIRY 156 10 SUMMARY AND CONCLUSIONS 1. The e f f e c t s of general anae s the t i c s were i n v e s t i g a t e d on pyramidal neurons of l a y e r s IV and V of guinea p i g neocortex i n i n v i t r o s l i c e p r epa r a t i o n s . The anae s the t i c a c t i o n s were examined, us ing i n t r a c e l l u l a r r eco rd ing techn iques , on pa s s i ve and a c t i v e membrane p r o p e r t i e s , the s ynap t i c t r a n s i e n t s evoked by orthodromic s t i m u l a t i o n , neuronal re spons i ve -ness to i o n t o p h o r e t i c a l l y a pp l i ed t r a n s m i t t e r substances and po s t sp i ke a f t e r h y p e r p o l a r i z i n g p o t e n t i a l s . 2. Two s t r u c t u r a l l y d i s s i m i l a r a nae s t he t i c s , i s o f l u r a n e - a halogenated e the r and A l t h e s i n - a s t e r o i d a l p r e p a r a t i o n , d i d not profoundly a f f e c t the pas s i ve membrane p r o p e r t i e s of the neurons, except at h igh doses. The h ighes t concen t ra t i on s used ( i s o f l u r a n e >2 MAC and A l t h e s i n >300 uM) h ype rpo l a r i z ed the neurons by 3-8 mV and inc reased t h e i r input conductance by 1 0 - 3 0 % . However, c l i n i c a l l y r e l e van t i nduc t i on doses of both agents produced a sharp, r e v e r s i b l e decrease i n the spontaneous a c t i v i t i e s of the neurons. The r e p e t i t i v e f i r i n g evoked by orthodromic s t i m u l a t i o n or by i n t r a c e l l u l a r c u r r e n t - p u l s e i n j e c t i o n s a l s o were s t r ong l y i n h i b i t e d by anae s the t i c a p p l i c a t i o n . 3. A dose-dependent, r e v e r s i b l e depress ion of n e o c o r t i c a l EPSPs was observed on a p p l i c a t i o n of i s o f l u r a n e and A l t h e s i n w i th E C 5 Q s of 1 MAC and ~50 uM r e s p e c t i v e l y . In order to e l i m i n a t e a p o s s i b l e shunt ing e f f e c t on EPSPs by the conductances i n vo l ved i n the concomitant IPSPs, a GABA A-an tagon i s t ( b i c u c u l l i n e ) was a p p l i e d together w i th the a n a e s t h e t i c s . A f t e r t h i s IPSP b lockade, the anae s the t i c s s t i l l depressed the EPSPs and were e f f e c t i v e i n reducing the e p i l e p t i f o r m a c t i v i t i e s evoked by b i c u c u l l i n e H. EL-BEHEIRY 1 5 7 dur ing subp ia l s t i m u l a t i o n . The IPSPs a l s o were evoked du r ing blockade of K-conductances by i n t e r n a l C s - a p p l i c a t i o n i n order t ha t the e f f e c t s of anaes the t i c s cou ld be s t ud i ed more a c cu r a te l y on the C l -med iated GABAergic IPSPs. A p p l i c a t i o n of i s o f l u r a n e or A l t h e s i n depressed these IPSPs i n a dose-dependent manner. 4 . D e n d r i t i c a p p l i c a t i o n s of a c e t y l c h o l i n e , g lutamate, NMDA and GABA d e p o l a r i z e d the pyramidal neurons. Somatic a p p l i c a t i o n of GABA induced only h y p e r p o l a r i z i n g responses t h a t were e i t h e r d e s e n s i t i z i n g or nonde sen s i t i z i n g and were l o n g - l a s t i n g i f the du ra t i on of GABA a p p l i c a t i o n was > 1 0 s. The responses i n l a y e r s IV and V neurons e x h i b i t e d t a chyphy l a x i s on f requent a p p l i c a t i o n of NMDA or GABA . 5 . I s o f l u r ane , A l t h e s i n and i n some cases, ha lothane, markedly reduced the d e p o l a r i z i n g responses and a s soc i a ted membrane conductance changes evoked by d e n d r i t i c a p p l i c a t i o n of a c e t y l c h o l i n e , g lutamate, and NMDA. The h y p e r p o l a r i z a t i o n evoked by GABA was not a f f e c t e d whereas the GABA evoked d e p o l a r i z a t i o n was depressed s l i g h t l y . The order of depres s i on was a c e t y l c h o l ine> glutamate and NMDA>» GABA. This s e l e c t i v i t y a l s o was assessed from the E C ^ Q f o r the i s o f l u r a n e - i n d u c e d depres s ion of the jus t -max imal responses to ACh which was 0 . 9 MAC compared w i t h the E C ^ Q of 1 . 9 MAC f o r the suppress ion of the g lutamate-responses. The s e l e c t i v i t y was l e s s pronounced i n the case of A l t h e s i n a c t i on s where the E C ^ s were 7 5 nM and 9 0 uM f o r the depres s ion of both t r a n s m i t t e r responses r e s p e c t i v e l y . 6. In order to i n v e s t i g a t e pa thophy s i o l o g i c a l mechanisms of anae s the t i c a c t i on s dur ing hypomagnesaemia, a c l i n i c a l c o n d i t i o n t h a t i s known to be a s s o c i a t ed w i t h inc reased anae s the t i c requ i rements , the a c t i o n s of anaesthe-t i c s were determined on the neuronal responses to p u t a t i v e t r a n s m i t t e r s H. EL-BEHEIRY 158 dur ing s imulated hypomagnesia i n v i t r o . The d e p o l a r i z a t i o n s evoked by a c e t y l c h o l i n e o r GABA were b locked by lower ing ex te rna l [Mg ] i n a decremental manner. Hypomagnesia reversed the e f f e c t s of i s o f l u r a n e and A l t h e s i n on the GABA and glutamate responses, i . e . , GABA ac t i on s were more depressed than those of g lutamate. 7. A s t r i k i n g ob se r va t i on i n t h i s study was the dose-dependent suppres-s i on by anae s the t i c s of the po s t sp i ke a f t e r h y p e r p o l a r i z a t i o n (AHP). The same e f f e c t was observed under c o n d i t i o n s of IPSP-blockade by b i c u c u l l i n e , which a l l e v i a t e d the AHPs (produced through a c t i v a t i o n of a Ca- a c t i v a t e d K-conductance) from the h y p e r p o l a r i z i n g , Cl-dependent i n f l u e n c e due to GABAergic a c t i v i t y . 8. Using Fura-2 m i c r o s p e c t r o f l u o r i m e t r y , the a c t i o n s of i s o f l u r a n e were 2+ s t ud i ed on g lu tamate- and K-evoked increases in [Ca ] . . The g lutamate 2+ evoked inc reases i n [Ca J . under c ond i t i o n s where a c t i o n s at NMDA- or q u i s q u a l a t e - r ecep to r s would be favoured, were at tenuated by i s o f l u r a n e . The v o l a t i l e anae s the t i c a l s o depressed the voltage-dependent inc reases i n 2+ [Ca ] . induced by e x t r a c e l l u l a r K a p p l i c a t i o n s under c o n d i t i o n s where Na-sp ike genes i s was b locked by TTX. 9. These i n v e s t i g a t i o n s p rov ide evidence tha t the depres s ion of neuronal e x c i t a b i l i t y i n neocortex induced by i s o f l u r a n e and A l t h e s i n i s due to a t t enua t i on of e x c i t a t i o n r a t he r than p o t e n t i a t i o n of i n h i b i t i o n . l t i s suggested t h a t these agents decrease the l e v e l of c o r t i c a l a c t i v i t y by i n t e r f e r i n g s p e c i f i c a l l y w i t h the ac t i on s of t r a n s m i t t e r s i n vo l ved i n mechanisms of behav ioura l a r o u s a l . The depress ion of the " i s o l a t e d " po s t -synapt i c Ca-conductance which i s c r u c i a l to the genera t i on of the AHPs together w i th a t t enua t i on s of the EPSPs, IPSPs and the g lutamate o r H. 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